Team:Freiburg Bioware/testpage

From 2010.igem.org

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===76.Labortag 01.08.2010===
+
===99. labday 24.08.2010===
-
====<p style="font-size:15px; background-color:#66bbFF;"><b>Picking clones of pSB1C3_CMV</b></p>====
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Sequence analysis of pSB1C3_CFP_middlelinker</b></p>====
-
<p><b>Investigator: Bea</b></p>
+
'''Investigator: Jessica'''
-
<p style="font-size:13px; color:#66bbff;"><b>Comments:</b> Trafo was performed friday, and trafo plate were incubated over night. Clones need to be picked in order to perform Mini-Prep</p>
+
<br>
-
<ul>
+
-
<li>Bacterial strain used: XL1-B</li>
+
-
<li>Two clones were picked of trafo plate</li>
+
-
<li>Inoculating of 10 mL DYT medium containing 10µL chloramphenicol</li>
+
-
<li> Put in 37°C room on rotary shaker</li>
+
-
</ul><br>
+
-
===77.Labortag 02.08.2010===
+
-
====<p style="font-size:15px; background-color:#66bbFF;"><b>Sequenzing of pSB1C3_RFC25_longlinker, pSB1C3_RFC25_SEG, pSB1C3_RFC25_GSAT </b></p>====
+
-
'''Investigator: Jessica'''<br>
+
-
<p style="font-size:13px; color:#66bbff;"><b>Comments:</b> Linkers (we got from Gerrit) we cloned in the RFC25-standard (pSB1C3_RFC25). Result looks good, linkers are in the vector pSb1C3_RFC25 <br>
+
-
<ul><li>pSB1C3_RFC25_longlinker '''P105 und P106'''</li>
+
-
<li>pSB1C3_RFC25_SEG '''P107 und P108'''</li>
+
-
<li>pSB1C3_RFC25_GSAT '''P109 und P110'''</li>
+
-
</ul>
+
-
 
+
-
</p>
+
-
 
+
-
[[File:Freiburg10_Sequence_alignment_longlinker.jpg|500px|left|thumb|]]
+
-
[[File:Freiburg10_Sequence_alignment_SEG.jpg|500px|left|thumb|]]
+
-
[[File:Freiburg10_Sequence_alignment_GSAT.jpg|500px|left|thumb|]]
+
-
<p style="clear:both;"> </p>
+
<br>
<br>
<br>
<br>
-
====<p style="font-size:15px; background-color:#FF00FF;"><b>Sequencing of pGA14_Prefix-leftITR</b></p>====
+
[[File:Freiburg10 pSB1C3_CFP_middlelinker.jpg|900px]]
-
'''Investigator: Hanna'''<br>
+
-
<br>
+
-
Eventually GATC partially managed to sequence pGA14_Prefix-left ITR clone 1 and 2. <br>
+
-
The sequencing results were better than before but in the ITR region still not evaluable due to the strong secondary structures. <br>
+
-
<br>
+
-
It seemed that, despite of successful insertion of the RFC10-Prefix, something went wrong because there were 2 bases missing in the NotI restriction site. But by having a closer look, it became obvious that the Geneious misinterpreted the chromatogram and overlaid the peaks of the missing bases. <br>
+
-
<br>
+
-
[[File:leftITR+Prefix.jpg|800px]]
+
-
<br>
+
-
<br>
+
-
For the first time sequencing of parts of the 3' end worked: The remaining NotI restriction site, which was already eliminated this week and also the "backbone-PstI" can be seen: <br>
+
-
<br>
+
-
[[File:leftITR+Prefix_2.jpg|800px]]
+
-
<br>
+
-
<br>
+
-
Sequencing of very GC-rich regions :) :) :) <br>
+
-
<br>
+
-
[[File:leftITR+Prefix_3.jpg|800px]]
+
-
<br>
+
-
<br>
+
-
Sequencing of clone 4 delivered that the ITR and the RFC10-Prefix were not inserted in the vector. The RFC25 standard of the multiple cloning site is still in the plasmid: <br>
+
-
<br>
+
-
[[File:LeftITR+Prefix clone4.jpg|800px]]
+
-
<br>
+
-
<br>
+
-
Therefore clone 4 was dicarded from the plasmid- and glycerol stock. <br>
+
<br>
<br>
 +
* '''pSB1C3_CFP_middlelinker is ready'''
-
====<p style="font-size:15px; background-color:#66bbFF;"><b>Repetition of test digestion pSB1C3_CFP SDM SspI and PvuII </b></p>====
 
-
'''Investigator: Jessica'''<br>
 
-
*buffer used: 2; Restriction-enzymes used: Enzyme 1: SspI ; Enzyme 2: PvuII
 
-
<ul><li>Plasmids (all: ~ 700 ng/µL):</li>
 
-
<ul><li>pSB1C3_CFP '''P51.1'''</li>
 
-
<li>pSB1C3_SDM_SspI '''P125'''</li>
 
-
<li>pSB1C3_SDM_PvuII '''P129'''</li>
 
-
<li>pSB1C3_SDM_PvuII '''P131'''</li></ul>
 
-
</ul>
 
-
<br />
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Harvest viral particles, Transduction of HT1080</b></p>====
-
{| border="1"
+
'''Investigator: Kerstin'''
-
| align="left" | '''Components'''  ||align="left"| '''Mastermix'''  ||align="left"| '''P125/µL''' ||align="left"| '''P51.1.1/µL''' ||align="left"| '''P129/µL''' ||align="left"| '''P131/µL''' ||align="left"| '''P51.1.2/µL'''
+
-
|-
+
-
| align="left" | DNA  ||align="left"| -||align="left"| 2,4||align="left"| 2,3||align="left"| 1,6||align="left"| 5,3||align="left"|5,3
+
-
|-
+
-
| align="left" | BSA (10x) ||align="left"| 6||align="left"| 1||align="left"| 1||align="left"| 1||align="left"| 1||align="left"| 1
+
-
|-
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-
| align="left" | Buffer 2 (10x)  ||align="left"| 6||align="left"| 1||align="left"| 1||align="left"| 1||align="left"| 12||align="left"| 1
+
-
|-
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-
| align="left" | Enzyme 1 SspI (68)  ||align="left"|-||align="left"| 1||align="left"| 1||align="left"| -||align="left"| -||align="left"| -
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-
|-
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| align="left" | Enzyme 2 PvuII (50)  ||align="left"| -||align="left"| -||align="left"| -||align="left"| 1||align="left"| 1||align="left"| 1
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-
|-
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-
| align="left" | H<sub>2</sub>O ||align="left"| -||align="left"| 4,6||align="left"| 4,7||align="left"| 5,4||align="left"| 1,7||align="left"| 1,7
+
-
|-
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-
| align="left" | '''Total volume''' ||align="left"| 12||align="left"| 10||align="left"| 10||align="left"| 10||align="left"| 10||align="left"| 10
+
-
|}
+
-
<br />
+
-
*Incubation: 65 minutes<br>
+
*Harvest viral particles following the standard protocol
-
'''Agarosegel'''
+
-
<br />
+
-
0.45 g Agarose, 50 ml TEB (0,5 %), 3 µL GELRED, at 115 Volt, running time: 45 minutes
+
-
<br />
+
-
<br />
+
-
<ul><li>Marker: GeneRuler ladder mix (6x)</li></ul>
+
*Transduction of three 6-well plates:
-
{| border="1"
+
-
|
+
-
!Marker
+
-
!P125
+
-
!P51.1 SspI
+
-
!-
+
-
!P129
+
-
!P131
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-
!P51.1 PvuII
+
-
|-
+
Plate 1 YFP: 150.000 cells per well<br />
-
!Lane
+
<table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
-
|7 µL
+
<tr>
-
|10 µL
+
<th width="80"> </th>
-
|10 µL
+
<th width="80">1</th>
-
| -
+
<th width="80">2</th>
-
|10 µL
+
<th width="80">3</th>
-
|10 µL
+
-
|10 µL
+
-
|-
+
</tr>
-
|}
+
<tr>
-
<br />
+
<td>A</td>
-
[[File:Freiburg10_Gel test digestion SDM.jpg|500px|left|thumb|]]
+
<td>control, no cells</td>
-
<br>
+
<td>500µl virus (1) </td>
-
<br>
+
<td>500µl virus (2) </td>
-
<br>
+
</tr>
-
<br>
+
<tr>
-
<br>
+
<td>B</td>
-
<br>
+
<td>control, no virus</td>
-
<br>
+
<td>500µl virus (1) </td>
-
<br>
+
<td>500µl virus (2) </td>
-
<br>
+
</tr>
-
<br>
+
</table>
-
<br>
+
Plate 2 YFP: 150.000 cells per well<br />
-
<br>
+
<table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
-
<br>
+
<tr>
-
<br>
+
<th width="80"> </th>
-
<br>
+
<th width="80">1</th>
-
<br>
+
<th width="80">2</th>
-
<br>
+
<th width="80">3</th>
-
<br>
+
-
<br>
+
-
<br>
+
-
<br>
+
-
<br>
+
-
<br>
+
-
<ul><li>'''P125''' isn't cut, therefore you see the bands for supercoiled, nicked and relaxed</li>
+
-
<li>'''P51.1''' is cut once, therefore you can see a faster running band</li>
+
-
<li>'''P129''' and '''P131''' should be cut once because of the PvuII in the CFP but there is another band we can't identify</li>
+
-
<li> '''P51.1''' should be cut two times but there is also another unidentified band</li>
+
-
<ul><li>perhaps there is another PvuII in the vector we can't find (???)</li></ul>
+
-
</ul>
+
-
<p style="font-size:13px; color:#66bbff;"><b>Comments:</b> SDM of SspI is ready! SDM of PvuII looks confusing because of the lower band. P 129 will be sequenced but this doesn't solve this problem. there has to be one more rs we don't know, perhaps. we will check it...anywise</p><br>
+
-
====<p style="font-size:15px; background-color:#66bbff;">'''Mini-Prep and Test digestion of pSB1C3_CMV  '''</p>====
+
</tr>
-
'''Investigator: Kerstin''' <br>
+
<tr>
-
'''Nanodrop'''
+
<td>A</td>
-
* pSB1C3_CMV clone1: 157,6 ng/µl '''P145'''
+
<td>control, no cells</td>
-
* pSB1C3_CMV clone2: 155,6 ng/µl '''P146'''
+
<td>500µl virus (3) </td>
-
<br>
+
<td>500µl virus (4) </td>
-
<br>
+
</tr>
-
'''Test digestion''' 900ng DNA
+
<tr>
-
{| border="1"
+
<td>B</td>
-
| components  || align="right" |P145 /µl || align="right" |P146 /µl
+
<td>control, no virus</td>
-
|-
+
<td>500µl virus (3) </td>
-
| DNA  ||  align="right" | 5,7||  align="right" |5,8
+
<td>500µl virus (4) </td>
-
|-
+
</tr>
-
| BSA (10x) ||  align="right" |1,5 ||  align="right" | 1,5
+
</table>
-
|-
+
Plate 3 YFP: 200.000 cells per well<br />
-
| Buffer 4 (10x)||  align="right" | 0,5 ||  align="right" | 0,5
+
<table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
-
|-
+
<tr>
-
| Enzyme 1: XbaI ||  align="right" | 0,5 ||  align="right" | 1,5
+
<th width="80"> </th>
-
|-
+
<th width="80">1</th>
-
| Enzyme 2: PstI ||  align="right" | 0,5 ||  align="right" | 0,5
+
<th width="80">2</th>
-
|-
+
<th width="80">3</th>
-
|H<sub>2</sub>O||  align="right" | 5,3||  align="right" | 5,2
+
-
|-
+
-
|'''Total volume (e.g. 15,20,25,30 µl)'''||  align="right" | '''15'''||  align="right" | '''15'''
+
-
|}
+
-
<br>
+
-
agarose gel: 1,5%, digestion: 90 minutes, 37°C
+
-
<br>
+
-
[[File:Freiburg 10 pSB1C3 CMV.jpg|400px|thumb|left|]]
+
-
<br>
+
-
<br>
+
-
<br>
+
-
<br>
+
-
<br>
+
-
<br>
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-
<br>
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<br>
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<br>
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-
<br>
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<br>
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<br>
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-
<br>
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<br>
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<br>
+
-
<br>
+
-
<br>
+
-
<br>
+
-
<br>
+
-
<br>
+
 +
</tr>
 +
<tr>
 +
<td>A</td>
 +
<td>control, no cells</td>
 +
<td>500µl virus (5) </td>
 +
<td>500µl virus (9) </td>
 +
</tr>
 +
<tr>
 +
<td>B</td>
 +
<td>control, no virus</td>
 +
<td> 500µl virus (9) </td>
 +
<td> 500µl virus (5) </td>
 +
</tr>
 +
</table>
 +
*(1)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,06
 +
*(2)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,08
 +
*(3)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,10
 +
*(4)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,12
 +
*(5)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,14
 +
*(9)= 10µg RC, 10µg pHelper, 3,3µg GOI (<b>eGFP</b>)
-
'''P145 is sent for sequencing with Reverse Primer (VR2)'''
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-prep of SDM NgoMIV CD</b></p>====
-
<p style="font-size:13px; color:#68bbff;">'''''Comments:'''''Results look good. CMV has the expected size (~ 662bp) </p><br>
+
'''Investigator: Kira''' <br />
-
'''</p>
+
Motivation: Harvest DNA for further analysis, e.g. test digestion
-
====<p style="font-size:15px; background-color:#66bbFF;"><b>Sent for sequencing</b></p>====
+
Mini-prep was performed on 3 colonies.
-
'''Investigator: Jessica'''<br>
+
 
-
'''Hanna'''<br>
+
c(clone1) = 358, 05 ng/ul
-
<ul><li>'''P145''' left ITR</li>
+
c(clone2) = 352, 10 ng/ul
-
<ul><li>Primer: GATC_std_pQE-FP and GATC_std_m13-FP</li></ul>
+
c(clone 3) = 376, 53 ng/ul
-
<li>'''P150''' right ITR</li>
+
-
<ul><li>Primer: GATC_std_pQE-FP and GATC_std_m13-FP</li></ul>
+
-
</ul>
+
-
<br>
+
-
'''Anissa'''<br>
+
-
<ul><li>'''P136''' pSB1C3_hgh</li>
+
-
<ul><li>Primer: O51_Reverse Primer (VR2)</li></ul>
+
-
<li>'''P139''' pAAV_BamHI</li>
+
-
<ul><li>Primer: O36_Rep_1250_rev</li></ul>
+
-
<li>'''P143''' pAAV_RC_SalI</li>
+
-
<ul><li>Primer: O53_Rep_1250_for</li></ul>
+
-
<li>'''P141''' pGL3_QTERT</li>
+
-
<ul><li>Primer: GATC_std_pTal-FP</li></ul>
+
-
<li>'''P133''' pSB1C3_betaGlobin</li>
+
-
<ul><li>Primer: O51_ReversePrimer (VR2)</li></ul>
+
-
<li>'''P145''' pSB1C3_CMV</li>
+
-
<ul><li>Primer: O51_ReversePrimer (VR2)</li></ul>
+
-
</ul>
+
-
<br>
+
-
'''Jessica'''<br>
+
-
<ul>
+
-
<li>'''P129''' pSB1C3_SDM_PvuII</li>
+
-
<ul>
+
-
<li>Primer: GATC_std_pTeSp-2</li>
+
-
</ul>
+
-
</ul>
+
-
<br>
+
-
====<p style="font-size:15px; background-color:#66bbFF;"><b>Preparation of competent E.coli</b></p>====
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Sent for sequencing: pSB1C3_6xHis</b></p>====
'''Investigator: Jessica'''
'''Investigator: Jessica'''
-
<ul><li>15ml DYT was prepared with 15µl tetracycline and inoculate with XL1B</li>
+
* Plasmid: '''P84''' c= 107,8 ng/µl
-
<li>10ml DYT w/o antibiotics was prepared and inoculate with BL21 (just for glycerol stock, no competent cells)</li></ul><br>
+
* Primer: VR2
-
both were incubate over night in 37°C room
+
* tube number: JG84
-
<br>
+
 
-
<br>
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Subcloning Cap into pAAV_RC_ins-rep</b></p>====
-
====<p style="font-size:15px; background-color:#FF00FF;"><b>Last Mini-Prep and test digestion of ITRs</b></p>====
+
'''Investigator: Stefan'''
-
'''Investigator: Hanna'''<br>
+
-
<br>
+
-
<p style="font-size:18px; font-weight: bold; color:#FF00FF;"><u>Plasmid Mini-Prep</u></p>
+
-
*new vector name: pGA14_RFC10_leftITR and pGA14_RFC10_rightITR
+
<br />
<br />
-
<u><b>Glycerol Stocks</b></u> <br>
 
-
<b>pGA14_RFC10_leftITR</b>
 
-
{| border="1"
 
-
| align="left" |  ||align="left"| '''Clone 1''' ||align="left"| '''Clone 2''' ||align="left"| '''Clone 3'''
 
-
|-
 
-
| align="left" | '''Bacteria strain''' ||align="left"| XL1blue ||align="left"| XL1blue  ||align="left"| XL1blue   
 
-
|-
 
-
| align="left" | '''Plasmidname''' ||align="left"| pGA14_RFC10_leftITR ||align="left"| pGA14_RFC10_leftITR ||align="left"| pGA14_RFC10_leftITR
 
-
|-
 
-
| align="left" | '''Date''' ||align="left"| 2.8.10 ||align="left"| 2.8.10 ||align="left"| 2.8.10 
 
-
|-
 
-
| align="left" | '''given glycerol-stock no.''' ||align="left"| B115 ||align="left"| B116 ||align="left"| B117
 
-
|-
 
-
| align="left" | '''given plasmid no.''' ||align="left"| P147 ||align="left"| P148 ||align="left"| P149 
 
-
|}
 
-
<br>
 
-
<b>pGA14_RFC10_rightITR</b>
+
Comment: Sequencing of pAAV_RC_final showed that Cap was not inserted succsessfully. Therefore it has to be repeated.
-
{| border="1"
+
-
| align="left" |  ||align="left"| '''Clone 1''' ||align="left"| '''Clone 2''' ||align="left"| '''Clone 3'''
+
-
|-
+
-
| align="left" | '''Bacteria strain''' ||align="left"| XL1blue ||align="left"| XL1blue  ||align="left"| XL1blue   
+
-
|-
+
-
| align="left" | '''Plasmidname''' ||align="left"| pGA14_RFC10_rightITR ||align="left"| pGA14_RFC10_rightITR ||align="left"| pGA14_RFC10_rightITR
+
-
|-
+
-
| align="left" | '''Date''' ||align="left"| 2.8.10 ||align="left"| 2.8.10 ||align="left"| 2.8.10 
+
-
|-
+
-
| align="left" | '''given glycerol-stock no.''' ||align="left"| B112 ||align="left"| B113 ||align="left"| B114
+
-
|-
+
-
| align="left" | '''given plasmid no.''' ||align="left"| P150 ||align="left"| P151 ||align="left"| P152 
+
-
|}
+
<br />
<br />
-
<p style="font-size:15px; font-weight: bold; color:#FF00FF;">Test digestion</p>
 
-
*buffer used: 4 ; Restriction-enzymes used: Enzyme 1 XbaI ; Enzyme 2 SpeI
 
-
*Plasmid: pGA14_RFC10_leftITR:
 
-
**Given Plasmid-Number: P147; DNA concentration: 240.22 ng/µL ;
 
-
**Given Plasmid-Number: P148; DNA concentration: 268.97 ng/µL ;
 
-
**Given Plasmid-Number: P149; DNA concentration: 234.16 ng/µL ;
 
-
<br>
 
-
*Plasmid: pGA14_RFC10_rightITR:
 
-
**Given Plasmid-Number: P150; DNA concentration: 221.71 ng/µL ;
 
-
**Given Plasmid-Number: P151; DNA concentration: 191.13 ng/µL ;
 
-
**Given Plasmid-Number: P152; DNA concentration: 215.14 ng/µL ;
 
<br />
<br />
-
 
+
'''Digestion of vector and insert:'''
 +
*Insert: pMA_RepCap Vector_SDM_InsPvuII clone 1 (P211)
 +
*Vector: pAAV_RC_ins-rep clone1 (P250)<br />
 +
<br />
 +
<br />
 +
A two-step digestion was performed: <br />
<br />
<br />
 +
'''1st step'''
{| border="1"
{| border="1"
-
| align="left" | '''Components''' ||align="left"| <b>P147</b> Volume/µL ||align="left"| <b>P150</b> Volume/µL
+
| || align="right" |'''P211 / µl''' ||'''P250 / µl'''
|-
|-
-
| align="left" | DNA ||align="left"| 4.2 ||align="left"| 4.5
+
|DNA|| align="right" |5,8  || align="right" | 5,2                             
|-
|-
-
| align="left" | BSA (10x) ||align="left"| 1 ||align="left"| 1
+
|buffer 3|| align="right" |2 || align="right" | 2
|-
|-
-
| align="left" | Buffer no. 4 (10x) ||align="left"| 1 ||align="left"| 1
+
| Enzyme BsiWI|| align="right" |1 || align="right" | 1
|-
|-
-
| align="left" | Enzyme 1 XbaI ||align="left"| 0.5 ||align="left"| 0.5
+
|H<sub>2</sub>O|| align="right" |11,2 || align="right" | 11,8
|-
|-
-
| align="left" | Enzyme 2 SpeI ||align="left"| 0.5 ||align="left"| 0.5
+
|total volume|| align="right" |20 || align="right" |20
-
|-
+
-
| align="left" | H<sub>2</sub>O ||align="left"| 2.8 ||align="left"| 2.5
+
-
|-
+
-
| align="left" | '''Total volume''' ||align="left"| <b>P147</b> ||align="left"| <b>P150</b>
+
|}
|}
<br />
<br />
-
 
+
'''2nd step'''
-
*Incubation: 1 h
+
-
<br />
+
-
<p style="font-size:15px; font-weight: bold; color:#FF00FF;">Agarose-Gel:</p>
+
-
<br />
+
-
0.83 g Agarose, 53 mL <b>TBE</b> (1.57%), 3 µL GELRED, at 115 Volt, running time: 45 minutes
+
-
<br />
+
-
<br />
+
-
{| border="1" cellspacing="0" cellpadding="2" bordercolor="black"
+
-
!Sample
+
-
!Sample/µl]
+
-
!Loading dye (5x)/µl
+
-
!Expected size 1 (Geneious)
+
-
!Expected size 2 (Geneious)
+
-
|--
+
-
|P147
+
-
|10 µl
+
-
|2 µl
+
-
|157 bp
+
-
|2902 bp
+
-
|--
+
-
|P150
+
-
|10 µl
+
-
|2 µl
+
-
|156 bp
+
-
|2903 bp
+
-
|--
+
-
 
+
-
|}
+
-
{| align=right
+
-
|}
+
-
<br />
+
-
*Marker: GeneRuler ladder mix
+
{| border="1"
{| border="1"
-
|
+
| || align="right" |'''P211 / µl''' ||'''P250 / µl'''
-
!Marker /µL
+
-
!Sample P147 /µL
+
-
!Sample P150 /µL
+
|-
|-
-
!Lane
+
|Mix obtained from step 1 || align="right" |20  ||  align="right" | 20                             
-
|6.5
+
-
|12
+
-
|12
+
|-
|-
-
|}
+
| Enzyme BspMI|| align="right" |1  || align="right" | 1
-
<br />
+
-
<br>
+
-
<p style="font-size:15px; font-weight: bold; color:#FF00FF;">Test digestion delivered positive results for both ITRs: The expected fragment sizes (156 resp. 157 bp) could be detected in the referring range between the 100 and 200 bp marker nucleotides:</p> <br>
+
-
[[File:LastITRPrep.jpg|400px]] <br>
+
-
<br>
+
-
<b>Comment:</b> In order to verify the results, P147 and P150 were sent for sequencing. Because sequencing never worked before, they will be sequenced under special conditions this time.<br>
+
-
<br>
+
-
 
+
-
====<p style="font-size:15px; background-color:#66bbff;">'''BioBrick production: mGMK in pSB1C3'''</p>====
+
-
Investigator: Stefan<br>
+
-
Backbone taken from: <br>
+
-
*pSB1C3_CFP (P51.2): 151,1 ng/µl<br>
+
-
Insert taken from: <br>
+
-
*pAAV_RFC25_mGMK (P100): 411,3 ng/µl<br>
+
-
 
+
-
 
+
-
 
+
-
{| border="1"
+
-
| components  || align="right" |pSB1C3_CFP(P51.2) /µl|| align="right" |pAAV_RFC25_mGMK (P100) /µl
+
|-
|-
-
| DNA  ||  align="right" |8 ||  align="right" | 5
+
|H<sub>2</sub>O||  align="right" |1,25 ||  align="right" | 1,25
|-
|-
-
| BSA (10x) ||  align="right" |2||  align="right" | 2
+
|total volume||  align="right" |22,25 ||  align="right" |22,25
-
|-
+
-
| Buffer 4 (10x)||  align="right" |2 ||  align="right" | 2
+
-
|-
+
-
|XbaI ||  align="right" |1 ||  align="right" | 1
+
-
|-
+
-
|AgeI ||  align="right" |1 ||  align="right" | 1
+
-
|-
+
-
|H<sub>2</sub>O||  align="right" |6 ||  align="right" | 9
+
-
|-
+
-
|'''Total volume '''||  align="right" |20 ||  align="right" | 20
+
|}
|}
-
<br>
 
-
 
-
* 1% Agarose gel
 
-
* 3 µl Gelred
 
-
*7 µl DNA-Ladder-Mix
 
-
* 115 Volt, running time: 50 minutes
 
-
Afer 50 minutes a picture (see below) was taken. Because there was no clear pattern (two bands) for the pSB1C3 backbone, the band of the insert mGMK was cut out and the gel ran another 20 minutes. After that, there were still two bands. Both were cut out and it was continued with two possible backbones. They were labeled vector (lower) and vector (upper).<br>
 
-
 
-
[[File:Freiburg10 BioBrick pSB1C3 mGMK.jpg|400px|thumb|left|]]<br>
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
 +
Samples were loaded on a 0,8% agarose gel an run at 115V for about 60 minutes.
 +
<gallery widths=400px heights=400px caption="pAAV RC final">
 +
Image:Freiburg10 Cap Insertion.png
 +
</gallery>
 +
'''Gelextraction:'''<br />
 +
Gelex was performed according to protocol exept for elution was done with 60 µl instead of 20 µl.<br />
 +
*c(P211)= 1,14 ng/µl
 +
*c(P250)= 5,60 ng/µl
<br />
<br />
-
 
-
{| border="1"
 
-
| '''Sample/µl ''' ||''' Expected size/bp
 
-
|-
 
-
| Vector pSB1C3  ||  align="right" |2082
 
-
|-
 
-
| Insert mGMK ||  align="right" |603
 
-
|-
 
-
|}
 
<br />
<br />
-
* weight of insert mGMK gel extract: 0,17 g
+
'''T4 Ligation:'''<br />
-
* weight of vector (lower) pSB1C3 gel extract: 0,08 g
+
T4 ligation was performed according to protocol.
-
* weight of vector (upper) pSB1C3 gel extract: 0,09 g
+
used DNA amounts: <br />
 +
*v(P211)= 2,56 µl
 +
*v(P250)= 5,44 µl
 +
<br />
 +
<br />
 +
'''Transformation:'''<br />
 +
<br />
 +
Trafo was performed according to protocol using XL1b cells.
-
'''Nanodrop'''
+
====<p style="font-size:15px; background-color:#66bbFF;"><b>Test digestion of pCerulean (p273) </b></p>====
-
* insert mGMK : 4,13 ng/µl
+
'''Investigator: Anissa'''<br>
-
* vector (lower) pSB1C: 5,47 ng/µl
+
-
* vector (upper) pSB1C: 0,60 ng/µl => for calculation of ligation approach calculated with 1 ng/µl
+
-
<br>
+
-
'''Ligation'''
+
<p style="font-size:13px; color:#66bbff;"><b>Comments:</b>Sequencing of pCerulean showed strange results, because the qualtity of sequenzing was not good. For working further with pCerulean, we started with a test digestion, to be sure, everything works.<br/>
-
*vector (lower) : insert - 4,16 µl : 4,84 µl
+
Because the sequencing showed no AgeI, we cut one time with AgeI and EcoRI, the other time with NotI.
-
*vector (upper) : insert - 7,43 µl : 1,57 µl<br>
+
</p>
-
 
+
<br />
-
'''Transformation'''
+
-
Two approaches were prepared, one for each vector and put in 37 °C room overnight.
+
-
<br>
+
-
<br>
+
-
 
+
-
====<p style="font-size:15px; background-color:#FF00FF;"><b>Sequencing results of SDM Rep 68 & 78</b></p>====
+
-
'''Investigator: Hanna and Volker'''<br>
+
-
The sequencing results delivered that the PstI restriction sites of Rep 68 and Rep 78 were successfully deleted via side-directed mutageneis:
+
-
<br>
+
-
<gallery widths=500px heights=300px perrow=2 caption="Sequencing results of SDM Rep 68 & 78">
+
-
File:Rep68.jpg |Rep 68
+
-
File:Rep78.jpg |Rep 78
+
-
</gallery>
+
-
 
+
-
===78.Labortag 03.08.2010===
+
-
====<p style="font-size:15px; background-color:#66bbff;">'''Results of sequencing pSB1C3_CFP_SDM_PvuII'''</p>====
+
-
'''Investigator: Jessica'''<br>
+
-
<b>SDM of PvuII:</b>
+
-
[[File:PvuII.jpg|600px]]
+
-
<br>
+
-
<br>
+
-
'''PvuII is succesfully deleted in the vector pSB1C3_RFC25_CFP'''
+
-
<br>
+
-
<br>
+
-
====<p style="font-size:15px; background-color:#66bbff;">'''Repetition of the quickchange site-directed mutagenesis of pAAV_RC_1.1_SalI'''</p>====
+
-
'''Investigator: Kerstin,Anissa'''<br>
+
-
<p style="font-size:13px; color:#68bbff;">'''''Comments:''''' Sequenzing revealed no mutagenesis of SalI in pAAV_RC_1.1_SalI, so SDM now will be repeated.</p> <br>
+
-
PCR-reaction:
+
{| border="1"
{| border="1"
-
| '''Volume / µl''' || align="right" |'''ingredients'''|| align="right" |'''recommended /µl'''
+
| align="left" | '''Components'''   ||align="left"| '''p273/µL''' ||align="left"| '''p273/µL'''  
-
|-
+
-
| 2,5  ||  align="right" |10X Pfu Ultra II buffer ||  align="right" | 2,5
+
|-
|-
-
| 4,18 ||  align="right" |template (~10 ng)|| align="right" | 4,18 of 1:100 dilution
+
| align="left" | DNA ||align="left"| 2||align="left"| 2
|-
|-
-
| 0,58|| align="right" |forward primer: O68|| align="right" | 62,5 ng
+
| align="left" | BSA (10x) ||align="left"|1,5||align="left"|1,5
|-
|-
-
|0,58 ||  align="right" |reverse primer: O69 || align="right" | 62,5 ng
+
| align="left" | Buffer 4 (10x) ||align="left"| 1,5||align="left"| 1,5
|-
|-
-
|- || align="right" |DMSO || align="right" | -
+
| align="left" | Enzyme 1 ||align="left"|0,5 NotI||align="left"|0,5 EcoRI
|-
|-
-
|0,5||  align="right" |dNTP|| align="right" | 250 µM each dNTP
+
| align="left" | Enzyme 2 ||align="left"|0,5 NotI ||align="left"|0,5 AgeI
|-
|-
-
|16,16||  align="right" |H<sub>2</sub>O || align="right" |
+
| align="left" | H<sub>2</sub>O ||align="left"| 9||align="left"| 9
|-
|-
-
|0,5||  align="right" |PfuUltra II fusion (1.25) || align="right" |
+
| align="left" | '''Total volume''' ||align="left"| 15||align="left"|15
|}
|}
-
<br>
+
<br />
-
PCR program:
+
[[Image:Freiburg10 pCerulean test digestion.jpg|200px|test digestion of pCerulean]]
-
{| border="1"
+
<br />
-
|'''Rounds'''|| align="right" |'''temperature/ °C'''|| align="right" |'''Time'''
+
<br />
-
|-
+
-
|1 ||  align="right" |95 ||  align="right" | 2 minutes
+
-
|-
+
-
| 20 ||  align="right" |95||  align="right" |30 seconds
+
-
|-
+
-
|20 ||  align="right" |80,4 ||  align="right" | 1 minute
+
-
|-
+
-
|20||  align="right" |68||  align="right" | 7,5 minutes
+
-
|}
+
-
<br>
+
-
Plasmid was transformed into BL21 cells. Clones have to be picked tomorrow.
+
-
<p style="font-size:13px; color:#ff0000;">'''''Comments:'''''NO CLONES, trafo didn't work, because annealing temperature was totally to high! (80,4 °C instead of 55°C) annealing temperature should always be 55°C (in case of troubleshooting temperature can be increased up to MAX. 68°C !)</p> <br>
+
-
====<p style="font-size:15px; background-color:#66bbff;">'''PCR for Biobrickproduction of Rep 40, 52, 68, 78 and APP'''</p>====
+
<p style="font-size:13px; color:#66bbff;"><b>Comments:</b> Results of digestion seem to be all right</p>
-
+
-
'''Investigators: Volker, Anna'''<br>
+
-
+
====<p style="font-size:15px; background-color:#66bbFF;"><b>Cloning of VP1up into PCerulean </b></p>====
-
Aim of the experiment:
+
'''Investigator: Anissa'''<br>
-
We wanted to produce biobricks from  the expression plasmids Rep40ex, Rep52 ex, the Rep 68 and 78 in which the PstI at position 310 was silenced and the AAP. This was performed by a PCR with primes that annealed to the ends of the later biobrick but also contained restriction sites that can be cut afterwards and cloned into pSB1C3.
+
VP1up was cut out of pSB1C3 and into pCerulean
-
 
+
<ul>
-
*Plasmids used as template:
+
<li> Digestion:
-
Rep_68_ex (p119):  c = 470,6 ng/µl <br>
+
<br /><br />
-
Rep_78_(p122):      c = 201,08 ng/µl <br>
+
-
Rep_40_(p22.2):    c = 673,1 ng/µl <br>
+
-
Rep_52_(p23.2):    c = 532,22 ng/µl <br>
+
-
pAAV-RC containing AAP ORF (p50):   c = 378,5 ng/µl <br>
+
-
 
+
-
 
+
-
 
+
-
*Primer used:
+
-
For Rep_40_ex: Praefix_40_52_ex & Suffix_40_68_ex<br>
+
-
For Rep_52_ex: Praefix_40_52_ex & Suffix_52_78_ex<br>
+
-
For Rep_68_ex: Praefix_68_78_ex & Suffix_40_68_ex<br>
+
-
For Rep_78_ex: Praefix_68_78_ex & Suffix_52_78_ex<br>
+
-
For AAP_ex: Praefix_AAP_ex & Suffix_AAP_ex
+
-
 
+
-
<br>
+
-
*'''PCR:
+
-
(was performed following the standard protocol)
+
-
<br>
+
{| border="1"
{| border="1"
-
| '''Ingredients''' || align="right" |'''Volume / µl''' || align="right" |'''Rep68'''|| align="right" |'''Rep78''' || align="right" |'''Rep40'''|| align="right" |'''Rep52'''|| align="right" |'''AAP'''
+
| align="left" | '''Components''' ||align="left"| '''Vector/µL''' ||align="left"| '''Insert/µL'''  
|-
|-
-
| 5X Phusion HF buffer ||  align="right" |10 || align="right" | ||  align="right" | || align="right" | ||  align="right" | ||  align="right" |  
+
| align="left" | DNA ||align="left"| 3,7||align="left"|8,4
|-
|-
-
| 10 mM dNTP mix||  align="right" |1|| align="right" | ||  align="right" | ||  align="right" | ||  align="right" | || align="right" |
+
| align="left" | BSA (10x) ||align="left"|1,5||align="left"|2
|-
|-
-
| forward primer: ||  align="right" |2,5|| align="right" | ||  align="right" | || align="right" | ||  align="right" | ||  align="right" |
+
| align="left" | Buffer 4 (10x) ||align="left"| 1,5||align="left"| 2
|-
|-
-
| reverse primer: ||  align="right" |2,5|| align="right" | ||  align="right" | || align="right" | ||  align="right" | ||  align="right" |
+
| align="left" | Enzyme 1 EcoRI ||align="left"|1||align="left"| 1
|-
|-
-
| DNA Template||  align="right" |***||  align="right" |4,2 µl || align="right" |1 µl || align="right" |2,9 µl||  align="right" |3,8 µl||  align="right" |5,3 µl
+
| align="left" | Enzyme 2 PstI  ||align="left"| 1||align="left"| 1
|-
|-
-
|- DMSO (2%)||  align="right" | ||  align="right" | ||  align="right" | ||  align="right" | || align="right" | || align="right" |
+
| align="left" | H<sub>2</sub>O ||align="left"|6,3||align="left"| 5,6
|-
|-
-
| Phusion Polymerase||  align="right" |0,5|| align="right" | ||  align="right" | || align="right" | ||  align="right" | ||  align="right" |
+
| align="left" | '''Total volume''' ||align="left"|15||align="left"| 20
-
|-
+
-
|H<sub>2</sub>O||  align="right" |*** ||  align="right" | 28,3 µl||  align="right" | 31,5 µl||  align="right" | 29,6 µl||  align="right" | 28,7 µl||  align="right" |27,2 µl
+
-
|-
+
-
|Total volume||  align="right" |50
+
|}
|}
-
<br>
+
<br />
-
 
+
</li>
-
PCR program:
+
<li> Gel:
 +
<br/>
 +
A 1% agarose-gel was made, after 30 minutes samples were cut<br/>
 +
[[Image:Freiburg10 Anissa24810.png|400px|test digestion of pCerulean]]
 +
<br/>
 +
</li>
 +
<li> Gelextraction was performed according the standardprotocol </li>
 +
<li> Ligation:
{| border="1"
{| border="1"
-
|'''PCR Program'''|| align="right" |'''temperature/ °C'''|| align="right" |'''Time''' || align="right" |'''Rep40'''|| align="right" |'''Rep50'''|| align="right" |'''Rep68'''|| align="right" |'''Rep78'''|| align="right" |'''AAP'''
+
| align="left" | '''Components''' ||align="left"| '''used volume for T4 ligation/µL''' ||align="left"| '''concentration /ng/µl'''  
|-
|-
-
|1||  align="right" |98 || align="right" |1min||  align="right" | || align="right" | ||  align="right" | ||  align="right" | ||  align="right" |
+
| align="left" | Vector ||align="left"| 5,15||align="left"|27,7
|-
|-
-
|2||  align="right" |98 || align="right" |15s||  align="right" | ||  align="right" | ||  align="right" | ||  align="right" | || align="right" |
+
| align="left" |Insert ||align="left"|2,85||align="left"|15,4
|-
|-
-
|8x||  align="right" |*** ||  align="right" |25s||  align="right" |63°C||  align="right" |62°C||  align="right" |63°C||  align="right" |62°C||  align="right" |64°C
 
-
|-
 
-
|3 ||  align="right" |72||  align="right" |***||  align="right" |15s  ||  align="right" |18s||  align="right" |24s ||  align="right" |27s ||  align="right" | 10s
 
-
|-
 
-
|4 ||  align="right" |98||  align="right" | 15s||  align="right" | ||  align="right" | ||  align="right" | ||  align="right" | ||  align="right" |
 
-
|-
 
-
|17x||  align="right" |***||  align="right" |25s||  align="right" |68°C||  align="right" |66°C||  align="right" |68°C||  align="right" |64°C||  align="right" |68°C
 
-
|-
 
-
|5||  align="right" |72||  align="right" |***||  align="right" |15s||  align="right" |18s||  align="right" |24s||  align="right" |27s||  align="right" |10s
 
-
|-
 
-
|6x||  align="right" |72||  align="right" |5min||  align="right" | ||  align="right" | ||  align="right" | ||  align="right" | ||  align="right" |
 
-
|-
 
-
|Hold||  align="right" |4||  align="right" |  ||  align="right" | ||  align="right" | ||  align="right" | ||  align="right" | ||  align="right" |
 
|}
|}
-
<br>
+
In addition 1µl T4-ligase and 1µl T4-ligase-buffer were added
 +
</li>
 +
<li>Transformation: was performes into Xl1 blue according the standard-protocol
 +
</li>
 +
<br />
 +
</ul>
 +
====<p style="font-size:15px; background-color:#66bbff;"><b>Sequence analysis of sequencing reads prepared yesterday (23.08.2010)</b></p>====
 +
<b>Investigator: Bea </b>
 +
<br />
 +
<p style="font-size:13px; color:#cc0033;"><I><b>GENERAL COMMENT:</b> pSB1C3 {NLS and VP1up} were sent for sequencing. Aswell as the pAAV_RC_final which contains all four mutations in the Rep-Cap gene and the integrated and synthesiezd rep and cap gene and the plasmid pCerulean whith the CMV promoter and sv40 polyadenylation site.</i> </p>
 +
<br />
 +
<p style="font-size:13px; color:#003399;"><b>Comments</b>: Sequence analysis of pAAV_RC_final containing the subcloned "rep" (ordered) and "Cap (ordered) plus the four mutations to delete the restriction sites PstI, BamHi and SalI. </p>
 +
<ul>
 +
<li>Primer used: </li>
 +
<ul>
 +
<li>VP1 primer for pKex</li>
 +
<li>GATC_std_SK</li>
 +
<li>Primer Cap 2800 rev</li>
 +
<li>Primer Cap 2800 for</li>
 +
<li>Primer Cap 3500 for</li>
 +
</ul>
 +
<li>Plasmid sequenced: P283 </li>
 +
<li>Sequence sample: iGEM4</li>
 +
<li>Stored in Geneious-folder: RepCap insert ordered</li>
 +
</ul>
-
Five µl of PCR-product were used for an analytical gel to see if the PCR worked. <br>
+
<gallery widths=400px heights=400px caption="pAAV RC final">
-
 
+
Image:Freiburg10 Seqanalysis pAAV RC final 2010 23 08.jpg
-
<gallery widths=500px heights=300px perrow=2 caption="BioBrick production for Rep and AAP">
+
-
Image:Freiburg10_Biobrick-production for Rep40-78ex and  AAPex.jpg|Alalytical gel
+
-
Image:Freiburg10_Biobrick-production for Rep40-78ex and  AAPex praeparative gel.jpg|Praeparative gel
+
</gallery>
</gallery>
 +
<br />
 +
<b>Results:</b> <p style="color:#00bbff;">Sequencing of the mutation ok. Rep integration worked. Cap integration needs to be repeated.</p>
-
<p style="color:red;">Comment: The result is that the PCR worked for the longer Rep proteins (68&78) with which a Quick-change was performed to remove the PstI(310) but not with the constructs for the smaller Rep variants (40&52) that were recieved from PD Kleinschmidt. As the longer Rep variants the AAP PCR Reaction also resulted in a PCR-product of the expected size. Unfortunately there were a secondary band of ~90bp for Rep_68_ex. For this reason we performed a praeparative gel and decided to cut the bands of all PCR products.</p> <br>
 
-
 
-
The bands marked in the gel picture were cut and a gel extraction was performed.
 
-
<p style="font-size:15px; font-weight: bold; color: blue;">Gel extraction</p>
 
<br />
<br />
-
Gel measurement:
+
<b>Next steps: Repetition of the integration of the synthesized cap gene performed by Stefan (see topic). </b>
 +
<br />
<br />
<br />
-
{| border="1"
 
-
| align="left" | '''Sample'''
 
-
| align="left" | '''Weight'''
 
-
| align="left" | '''Volume'''
 
-
| align="left" | '''Concentration'''
 
-
|-
 
-
| align="left" | Rep_68_ex
 
-
| align="left" | 0,1 g
 
-
| align="left" | 20 µl
 
-
| align="left" | 17,6 ng/µl
 
-
|-
 
-
| align="left" | Rep_78_ex
 
-
| align="left" | 0,15g
 
-
| align="left" | 20 µl
 
-
| align="left" | 80,6 ng/µl
 
-
|-
 
-
| align="left" | AAP_ex
 
-
| align="left" | 0,19 g
 
-
| align="left" | 20µl
 
-
| align="left" | 77,78 ng/µl
 
-
|}<br>
 
-
====<p style="font-size:15px; background-color:#66bbFF;"><b>Continuation of preparation of competent E.coli</b></p>====
+
<p style="font-size:13px; color:#003399;"><b>Comments</b>: Sequence analysis of pSB1C3_NLS </p>  
-
'''Investigator: Jessica'''<br>
+
-
preparation of competent XL1blue was finished according to the standard protocol
+
-
<ul><li>aliquots of 60µl (to use for 1 trafo) are stored in -80°C freezer</li></ul>
+
-
 
+
-
====<p style="font-size:15px; background-color:#66bbFF;"><b>Sequencing of pSB1C3</b></p>====
+
-
'''Investigator: Jessica'''<br>
+
-
<p style="font-size:13px; color:#68bbff;">'''''Comments:''''' We sent for sequencing  '''pSB1C3_RFC25_longlinker (P105)''' to check the strange result of the test digestion of pSB1C3_SDM_PvuII(01.08.10. <br>
+
-
pSB1C3_SDM_PvuII is too long for sequencing wherefore we sent pSB1C3_RFC25_longlinker because the longlinker just have 54kb (bp, nicht kb (Volker) (necessary is just the backbone we don't have.</p>
+
-
<ul><li>Primer:</li>
+
<ul>
<ul>
-
<li>RESgen-241698</li>
+
<li>Primer used: VR-2</li>
-
<li>Reverse primer (VR2)</li>
+
<li>Plasmid sequenced: P282 </li>
-
<li>o_F1-fw (from Gerrit)</li>
+
<li>Sequence sample: iGEM3_O51_VR-2 </li>
 +
<li>Stored in folder: pSB1C3_NLS</li>
</ul>
</ul>
-
</ul>
 
-
<br>
 
-
<br>
 
-
====<p style="font-size:15px; background-color:#FF00FF;"><b>Design of Primer for p5 Promoter WT and TATA-less BioBrick production</b></p>====
+
<gallery widths=600px heights=400px caption="pSB1C3_NLS">
-
'''Investigator: Hanna'''<br>
+
Image: Freiburg10 Sequence analysis pSB1C3 NLS 2010 23 08.jpg
-
<br>
+
</gallery>
-
[[File:Freiburg10 p5 Primer.JPG|500px|thumb|left|]]
+
<br />
-
<br>
+
<b>Results:</b> <p style="color:#00bbff;">Insertion of the nuclear localisation signal (NLS) into the pSB1C3_NLS as one step in the N-terminal insertion of targeting molecules. Sequencing results look good. prefix and suffix ok!</p>
-
 
+
-
[[File:Freiburg10 p5Primer Aim.JPG|500px|thumb|right|]]
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+
-
===79.Labortag 04.08.2010===
+
-
 
+
-
====<p style="font-size:15px; background-color:#66bbff;">'''Cloning of right ITR and left ITR into pSB1C3_RFC25_CFP'''</p>====
+
-
Intention: Get biobricks ready. <br>
+
-
Investigator: Patrick <br>
+
-
pGA14_lITR_RFC10 (P147), pGA14_rightITR (P150) and pSB1C3_RFC25_CFP (P51.1) were digested with EcoRI and PstI (Buffer 4) according to the standard protocol. Digestion Time: 80 minutes.
+
-
<br>
+
-
GelRun: 1% Agarose Gel. Expected results (from left to right):
+
-
*pSB1C3_RFC25_CFP: about 2100 bp and 800 bp.
+
-
*pGA14_leftITR_RFC10: the size of the insert should be 135 bp.
+
-
*pGA14_rightITR_RFC10: the size of the insert should be 138bp.
+
-
[[File:20100704_patrick_bearbeitet.JPG|400px]]<br>
+
-
<br>
+
-
The mutual vector (now without CFP) and inserts (left ITR and right ITR) were cut out. The gelextraction was performed according to the standard protocol. DNA concentration of the extracts:
+
-
* pSB1C3_RFC25_CFP: 2,9 ng/µl
+
-
* left ITR: 1,4 ng/µl
+
-
* right ITR: 2,0 ng/µl
+
-
<br>
+
-
The Quick Ligation was not performed according to the standard protocol:
+
-
* left ITR + pSB1C3: 5µl Buffer, 1 µl Quick-Ligase, 2,5 µl left ITR, 1,5 µl pSB1C3_RFC25.
+
-
* right ITR + pSB1C3: 5µl Buffer, 1 µl Quick-Ligase, 2,5 µl right ITR, 1,5 µl pSB1C3_RFC25.
+
-
 
+
-
<br>
+
-
Transformation: performed according to the standard protocol (BL21). The cells were plated on a agar plate with chloramphenicol. The clones will be picked tomorrow.
+
-
<br>
+
-
 
+
-
====<p style="font-size:15px; background-color:#66bbff;">'''Cloning of SDM SspI and SDM PvuII'''</p>====
+
-
'''Investigator: Jessica'''
+
-
*Vector: name: pSB1C3_SDM_SspI '''P125'''
+
-
*Insert: name: pSB1C3_SDM_PvuII '''P129'''
+
-
*new vector name: pSB1c3_SDM_SspI/PvuII '''P157'''
+
-
*buffer used: 4 ; Restriction-enzymes used: Enzyme 1 (no. Lab: 141) BstI ; Enzyme 2 (no.Lab: 144) BpmI
+
-
*DNA concentration (vector): 321,9 ng/µl ; DNA concentration (insert): 308,4 µg/µl
+
<br />
<br />
-
{| border="1"
+
<b>Next steps: Fuse NLS to targeting molecule. </b>
-
| '''components'''  || align="right" |'''volume of pSB1C3_SDM_SspI /µl''' || align="right" |'''volume of pSB1C3_SDm_PvuII /µl'''
+
<br />
-
|-
+
<br />
-
| DNA  ||  align="right" |4,66 ||  align="right" |4,87
+
<p style="font-size:13px; color:#003399;"><b>Comments</b>: Sequence analysis of pSB1C3_VP1up</p>
-
|-
+
<ul>
-
| BSA (10x) ||  align="right" |2 ||  align="right" | 2
+
<li>Primer used: VF-2</li>
-
|-
+
<li>Plasmid sequenced: P280 </li>
-
| Buffer 4 (10x)||  align="right" |2 ||  align="right" |2
+
<li>Sequence sample: iGEM1_VF-2 </li>
-
|-
+
<li>Stored in folder: N-Terminal targeting --> pSB1C3_VP1up</li>
-
|Enzyme BstI (no.Lab:141)||  align="right" |1 ||  align="right" |1
+
</ul>
-
|-
+
<gallery widths=600px heights=400px caption="pSB1C3_VP1up">
-
|Enzyme BpmI (no.Lab:144)||  align="right" |1 ||  align="right" |1
+
Image:Freiburg10 Seqanalysis pSB1C3 VP1up 2010 23 08.jpg
-
|-
+
</gallery>
-
|H2O||  align="right" |9,34 || align="right" |9,13
+
<br />
-
|-
+
<b>Results:</b> <p style="color:#00bbff;"> Prefix ok, but annotation is wrong. VP1up should start with: GC!! Suffix ok! The additional EcoRI before the prefix standard can be due to the bad sequence read quality.</p>
-
|'''Total volume (e.g. 15,20,25,30 µl)'''||  align="right" | 20||  align="right" |20
+
-
|}
+
<br />
<br />
-
0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 130 Volt, running time:45
+
<b>Next steps: Clone VP1up into pCerulean in order to obtain the plasmid with a CMV promoter and the sv40 polyadenylation site. </b>
<br />
<br />
<br />
<br />
 +
<p style="font-size:13px; color:#003399;"><b>Comments</b>: Sequence analysis of pCerulean </p>
 +
<ul>
 +
<li>Primer used: GATC_CMV-F</li>
 +
<li>Plasmid sequenced: P273 </li>
 +
<li>Sequence sample: iGEM2_CMV-F </li>
 +
<li>Stored in folder: N-Terminal targeting --> pCerulean</li>
 +
</ul>
 +
<gallery widths=600px heights=400px caption="pCerulean">
 +
Image:Freiburg10 Seqanalysis pCerulean 2010 23 08.jpg
 +
</gallery>
 +
<br />
 +
<b>Results:</b> <p style="color:#00bbff;"> It seem that the suffix is not correct which means that AgeI, PstI and NotI is missing. This can be due to the bad sequence quality. Prefix is ok!
 +
</p>
<br />
<br />
-
'''Loading plan for agarose gel''': <br>
+
<b>Next steps: For verification a test digestion with the missing restriction sites will be perforemd and another round of sequencing will be conducted. </b>
-
Marker used: GeneRuler ladder mix (Fermentas)<br>
+
<br />
 +
<br />
-
{| border="1"
+
[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]<br />
-
|
+
-
!Marker
+
-
!Sample 125, 20µl
+
-
!Sample 129, 20µl
+
-
|-
+
-
!Lane
+
-
|1
+
-
|3
+
-
|5
+
-
|-
+
-
|}
+
-
<br>
+
-
'''Results''': digesttemperature of BstI is 55°C, plasmid was just digested at 37°C... :( (<br>
+
-
[[File:Digestion of SDM SspI and SDM PvuII.jpg|400px]] <br>
+
-
====<p style="font-size:15px; background-color:#66bbff;">'''2nd Repetition of Quickchange site-directed mutagenesis of pAAV_RC_1.1_SalI'''</p>====
+
====<p style="font-size:15px; background-color:#66bbFF;"><b>Test digestion of SDM NgoMIV CD </b></p>====
-
'''Investigator: Kerstin, Anissa'''<br>
+
'''Investigator: Kira'''<br>
-
<p style="font-size:13px; color:#68bbff;">'''''Comments:''''' Last trafo didn't work. Annealing temperature was to high (80,4°C instead of 55°C) </p> <br>
+
Motivation: In order to check if site directed mutagenesis was successfull, test digestion was performed with 3 clones, which were picked yesterday as well as with 'original' DNA, which still contains all the restriction sites
-
Two approaches were made (short and long PCR:
+
<li> Test digestion:
-
+
<br /><br />
-
PCR-reaction:
+
{| border="1"
{| border="1"
-
| '''Volume / µl''' || align="right" |'''ingredients'''|| align="right" |'''recommended /µl'''
+
| align="left" | '''Components''' ||align="left"| '''clone 1/µL''' ||align="left"| '''clone 2/µL''' ||align="left"| '''clone 3/µL'''||align="left"| '''original/µL'''
|-
|-
-
| 2,5 || align="right" |10X Pfu Ultra II buffer || align="right" | 2,5
+
| align="left" | DNA (500ng) ||align="left"| 1,4||align="left"|1,4||align="left"| 1,4||align="left"| 1,4
|-
|-
-
| 1,83 || align="right" |template (~10 ng): p139 (c: 546,97 ng/µl)|| align="right" | 1,83µl of 1:100 dilution
+
| align="left" | BSA (10x) ||align="left"| 1||align="left"| 1||align="left"| 1||align="left"| 1
|-
|-
-
| 0,58||  align="right" |forward primer: O68|| align="right" | 62,5 ng
+
| align="left" | Buffer 4 (10x) ||align="left"| 1||align="left"| 1||align="left"| 1||align="left"| 1
|-
|-
-
|0,58 || align="right" |reverse primer: O69 || align="right" | 62,5 ng
+
| align="left" | Enzyme 1 NgoMIV ||align="left"|0,5||align="left"| 0,5||align="left"|0,5||align="left"|0,5
|-
|-
-
|- || align="right" |DMSO || align="right" | -
+
| align="left" | H<sub>2</sub>O ||align="left"|6,1||align="left"| 6,1||align="left"| 6,1||align="left"| 6,1
|-
|-
-
|0,5||  align="right" |dNTP|| align="right" | 250 µM each dNTP
+
| align="left" | '''Total volume''' ||align="left"|10||align="left"| 10||align="left"| 10||align="left"| 10
-
|-
+
-
|18,51||  align="right" |H<sub>2</sub>O || align="right" |
+
-
|-
+
-
|0,5||  align="right" |PfuUltra II fusion (1.25) || align="right" |
+
|}
|}
-
<br>
 
-
PCR program (long):
 
-
{| border="1"
 
-
|'''Cycles'''|| align="right" |'''temperature/ °C'''|| align="right" |'''Time'''
 
-
|-
 
-
|1 ||  align="right" |95 ||  align="right" | 2 minutes
 
-
|-
 
-
| 20 ||  align="right" |95||  align="right" |30 seconds
 
-
|-
 
-
|20 ||  align="right" |55 ||  align="right" | 1 minute
 
-
|-
 
-
|20||  align="right" |68||  align="right" | 7,5 minutes
 
-
|}
 
-
<br>
 
-
PCR program (short):
 
-
{| border="1"
 
-
|'''Cycles'''|| align="right" |'''temperature/ °C'''|| align="right" |'''Time'''
 
-
|-
 
-
|1 ||  align="right" |95 ||  align="right" | 2 minutes
 
-
|-
 
-
| 20 ||  align="right" |95||  align="right" |30 seconds
 
-
|-
 
-
|20 ||  align="right" |55 ||  align="right" | 1 minute
 
-
|-
 
-
|20||  align="right" |68||  align="right" | 4 minutes
 
-
|}
 
-
Plasmids were transformed into BL21 cells. Clones have to be picked tomorrow.
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-Prep of pSB1C3_mGMK</b></p>====
 
-
<b>Investigator: Chris W., Bea</b><br>
 
-
 
-
 
-
Vector name: pSB1C3_mGMK upper band 1.1 and pSB1C3_mGMK upper band 1.2 and pSB1C3_mGMK lower band 2.1 and pSB1C3_mGMK lower band 2.2
 
-
 
-
Mini-Prep following the standart Protokoll
 
<br />
<br />
-
<li>P153 = pSB1C3_mGMK upper band 1.1 = 248,3 ng/µl
+
-
<li>P154 = pSB1C3_mGMK upper band 1.2 = 251,6 ng/µl
+
-
<li>P155 = pSB1C3_mGMK lower band 1.1 = 263 ng/µl
+
-
<li>P156 = pSB1C3_mGMK lower band 1.2 = 235,2 ng/µl
+
<br />
<br />
-
<br>
+
[[image:Freiburg10_Kopfkratzen.gif|thumb|right| Bitte schneid mich aus !]]
-
<br>
+
-
<b>Test digestion:</b>
+
-
<ul>
+
-
<li>Perfomed with 15 µL of total volume with all four clones</li>
+
-
<table border=1 cellpadding=0 cellspacing=0 width=621 style='border-collapse:
+
-
collapse;table-layout:fixed;width:466pt'>
+
-
<tr height=29 style='mso-height-source:userset;height:21.75pt'>
+
-
  <td height=29 class=xl6724191 width=143 style='height:21.75pt;width:107pt'>&nbsp;</td>
+
-
  <td class=xl6824191 width=131 style='border-left:none;width:98pt'>Mastermix/µL</td>
+
-
  <td class=xl6824191 width=87 style='border-left:none;width:65pt'>P153/µL</td>
+
-
  <td class=xl6824191 width=84 style='border-left:none;width:63pt'>P154/µL</td>
+
-
  <td class=xl6824191 width=90 style='border-left:none;width:68pt'>P155/µL</td>
+
-
  <td class=xl6824191 width=86 style='border-left:none;width:65pt'>P156/µL</td>
+
-
</tr>
+
-
<tr height=29 style='mso-height-source:userset;height:21.75pt'>
+
-
  <td height=29 class=xl6624191 width=143 style='height:21.75pt;border-top:
+
-
  none;width:107pt'><span lang=EN-US style='mso-ansi-language:EN-US'>DNA</span></td>
+
[[file:Freiburg10 test digestion NgoMIV.jpg]]
-
  <td class=xl6524191 style='border-top:none;border-left:none'>-</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>3,5</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>3,5</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>3,5</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>3,5</td>
+
-
</tr>
+
-
<tr height=29 style='mso-height-source:userset;height:21.75pt;mso-yfti-irow:
+
-
  1'>
+
According to the gel, NgoMIV SDM was successful in clones 1 and 2. Clone 3 well reveales 2 bands of unknown origin. Samples 1 and 3 were sent for sequencing.
-
  <td height=29 class=xl6624191 width=143 style='height:21.75pt;border-top:
+
-
  none;width:107pt'><span lang=EN-US style='mso-ansi-language:EN-US'>BSA (10x)</span></td>
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Picking clones of pSB1C3_Affibody_linker and pSB1C3_ß-Globin_YFP_hGH_rITR  </b></p>====
-
  <td class=xl6524191 style='border-top:none;border-left:none'>7,5</td>  
+
<b>Investigator: Anna </b>
-
  <td rowspan=4 class=xl6524191 style='border-top:none'>4</td>  
+
-
  <td rowspan=4 class=xl6524191 style='border-top:none'>4</td>  
+
-
  <td rowspan=4 class=xl6524191 style='border-top:none'>4</td>  
+
-
  <td rowspan=4 class=xl6524191 style='border-top:none'>4</td>  
+
-
</tr>
+
-
<tr height=29 style='mso-height-source:userset;height:21.75pt;mso-yfti-irow:
+
-
  2'>
+
<p style="font-size:13px; color:#003399;"><b>Comments</b>: A 1:1000 dilution of pSB1C3_Affibody_GSAT-Linker was prepared. <br/>
-
  <td height=29 class=xl6624191 width=143 style='height:21.75pt;border-top:
+
<b>To do</b>: Mini-Prep of pSB1C3_Affibody_(Short/Middle/Long and SEG-Linker) and pSB1C3_ß-Globin_YFP_hGH_rITR. Picking clones of pSB1C3_Affibody_GSAT-Linker. </p>
-
  none;width:107pt'><span lang=EN-US style='mso-ansi-language:EN-US'>Buffer
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Test digestion of pSB1C3_SDM_SspI_Bla14FM (P222), pSB1C3_BLA (P286, P288)  </b></p>====
-
  4<span style='mso-spacerun:yes'>   </span>(10x)</span></td>  
+
<b>Investigator: Achim</b>
-
  <td class=xl6524191 style='border-top:none;border-left:none'>7,5</td>  
+
-
</tr>  
+
-
<tr height=29 style='mso-height-source:userset;height:21.75pt;mso-yfti-irow:
+
-
  3'>
+
<p style="font-size:13px; color:#003399;"><b>Comments</b>New test digestion of two SDM-attempts, this time we also digested the original vector containing the PvuII restriction site to see differences.</p>
-
  <td height=29 class=xl6624191 width=143 style='height:21.75pt;border-top:
+
<br/>
-
  none;width:107pt'><span lang=EN-US style='mso-ansi-language:EN-US'>Enzyme
 
-
  XbaI<span style='mso-spacerun:yes'> </span></span></td>
 
-
  <td class=xl6524191 style='border-top:none;border-left:none'>2,5</td>
 
-
</tr>
 
-
<tr height=29 style='mso-height-source:userset;height:21.75pt;mso-yfti-irow:
 
-
  4'>
+
[[File:Freiburg10 Test digestion of pSB1C3 BLA final 24 08 2010.jpg|800px]]
-
  <td height=29 class=xl6624191 width=143 style='height:21.75pt;border-top:
+
-
  none;width:107pt'><span lang=EN-US style='mso-ansi-language:EN-US'>Enzyme
 
-
  AgeI</span></td>
 
-
  <td class=xl6524191 style='border-top:none;border-left:none'>2,5</td>
 
-
</tr>
 
-
<tr height=29 style='mso-height-source:userset;height:21.75pt;mso-yfti-irow:
 
-
  5;mso-yfti-lastrow:yes'>
 
-
  <td height=29 class=xl6624191 width=143 style='height:21.75pt;border-top:
 
-
  none;width:107pt'><span lang=EN-US style='mso-ansi-language:EN-US'>H2O</span></td>
+
*The original vector is being cut as expected: the uncut vector can be seen in different conformations, the linearized vector shows one distinct band and the digestions cutting out BLA show a band at ~900 bp. Our SDM attempts show inconsistent results, not only does the Pvu site still seem to be in the backbone, there are no more proper insert bands visible either. We therefore conclude that either the DpnI digestion or the dilution of the transformation must have gone wrong (differing plasmids...). Tomorow we'll try a new cloning approach using an old vector with deleted Pvu restriction site and MscI & XbaI digestion. In case this also fails we also yet have to prep and test digest the repetition of the mutagenesis which was carried out by Stefan.
-
  <td class=xl6524191 style='border-top:none;border-left:none'>-</td>
+
 
-
  <td class=xl6524191 style='border-top:none;border-left:none'>7,5</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>7,5</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>7,5</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>7,5</td>
+
-
</tr>
+
-
<tr height=29 style='mso-height-source:userset;height:21.75pt'>
+
-
  <td height=29 class=xl6624191 width=143 style='height:21.75pt;border-top:
+
-
  none;width:107pt'>Total volume</td>
 
-
  <td class=xl6524191 style='border-top:none;border-left:none'>15</td>
 
-
  <td class=xl6524191 style='border-top:none;border-left:none'>15</td>
 
-
  <td class=xl6524191 style='border-top:none;border-left:none'>15</td>
 
-
  <td class=xl6524191 style='border-top:none;border-left:none'>15</td>
 
-
  <td class=xl6524191 style='border-top:none;border-left:none'>15</td>
 
-
</tr> 
 
-
<tr height=0 style='display:none'>
 
-
  <td width=143 style='width:107pt'></td>
 
-
  <td width=131 style='width:98pt'></td>
 
-
  <td width=87 style='width:65pt'></td>
 
-
  <td width=84 style='width:63pt'></td>
 
-
  <td width=90 style='width:68pt'></td>
 
-
  <td width=86 style='width:65pt'></td>
 
-
</tr>
 
-
</table>
 
-
<br />
 
-
</ul>
 
-
<ul>
 
-
<li>All four samples were loaded on a 1% agarose gel</li>
 
-
<li>P153 = pSB1C3_mGMK upper band 1.1 </li>
 
-
<li>P154 = pSB1C3_mGMK upper band 1.2 </li>
 
-
<li>P155 = pSB1C3_mGMK lower band 1.1</li>
 
-
<li>P156 = pSB1C3_mGMK lower band 1.2</li>
 
</ul>
</ul>
 +
<gallery widths=600px heights=400px caption="pSB1C3_VP1up">
 +
Image:Freiburg10 Seqanalysis pSB1C3 VP1up 2010 23 08.jpg
 +
</gallery>
<br />
<br />
-
[[File:Freiburg10 04 08 2010 Test digestion of pSB1C3 mGMK.jpg|400px]]
+
<b>Results:</b> <p style="color:#00bbff;"> Prefix ok, but annotation is wrong. VP1up should start with: GC!!  Suffix ok! The additional EcoRI before the prefix standard can be due to the bad sequence read quality.</p>
 +
 
<br />
<br />
 +
<b>Next steps: Clone VP1up into pCerulean in order to obtain the plasmid with a CMV promoter and the sv40 polyadenylation site. </b>
<br />
<br />
<br />
<br />
-
<b> Sequencing</b>:  
+
<p style="font-size:13px; color:#003399;"><b>Comments</b>: Sequence analysis of pCerulean </p>
<ul>
<ul>
-
<li> Plasmid used: P156: pSB1C3_mGMK lower band 1.2</li>
+
<li>Primer used: GATC_CMV-F</li>
-
<li> Primer used: VR-2</li>
+
<li>Plasmid sequenced: P273 </li>
-
<li> Tube name: Bea_1</li>
+
<li>Sequence sample: iGEM2_CMV-F </li>
 +
<li>Stored in folder: N-Terminal targeting --> pCerulean</li>
</ul>
</ul>
 +
<gallery widths=600px heights=400px caption="pCerulean">
 +
Image:Freiburg10 Seqanalysis pCerulean 2010 23 08.jpg
 +
</gallery>
 +
<br />
 +
<b>Results:</b> <p style="color:#00bbff;"> It seem that the suffix is not correct which means that AgeI, PstI and NotI is missing. This can be due to the bad sequence quality. Prefix is ok!
 +
</p>
-
====<p style="font-size:15px; background-color:#66bbff;">''' cell culture '''</p>====
 
-
 
-
Investigator: Adrian
 
<br />
<br />
-
<b> Plan for the next virus production procedures </b>
+
<b>Next steps: For verification a test digestion with the missing restriction sites will be perforemd and another round of sequencing will be conducted. </b>  
<br />
<br />
-
The Motivation: investigation of the influence of different GOI amounts.
 
-
<br />
 
-
The Plan: keep rep/cap and pHelper amount stable (3,3 µg each => 6,6 µg), differ the GOI (YFP) amount in each stock.
 
-
<br />
 
-
Viral Stocks:
 
-
* 3,3 µg YFP + 3,3 µg pHelper + 3,3  rep/cap
 
-
* 10 µg YFP + 3,3 µg pHelper + 3,3  rep/cap
 
-
* 20 µg YFP + 3,3 µg pHelper + 3,3  rep/cap
 
-
<br />
 
-
*3,3 µg pHelper + 3,3  rep/cap (<b>without GOI !!!</b>)
 
-
<br />
 
-
*one GMK_TK clone (with the confirmed sequence) + 3,3 µg pHelper + 3,3  rep/cap
 
<br />
<br />
-
<b>Transduction plan</b>
+
[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]<br />
-
<li>five 6-well-plates wille be transduced
+
====<p style="font-size:15px; background-color:#66bbFF;"><b>Test digestion of SDM NgoMIV CD </b></p>====
-
{| align=right
+
'''Investigator: Kira'''<br>
-
|}
+
Motivation: In order to check if site directed mutagenesis was successfull, test digestion was performed with 3 clones, which were picked yesterday as well as with 'original' DNA, which still contains all the restriction sites
-
{| border="1"
+
-
|A
+
-
!150µl AAV stock 1
+
-
!300µl AAV stock 1
+
-
!control no Virus
+
-
|-
+
-
!B
+
-
|150µl AAV stock 2
+
-
|300µl AAV stock 2
+
-
|150µl AAV without GOI
+
-
|-
+
-
|}
+
-
====<p style="font-size:15px; background-color:#66bbff;">'''Repetition of cloning of SDM SspI and SDM PvuII'''</p>====
+
<li> Test digestion:
-
'''Investigator: Jessica'''
+
<br /><br />
-
*Vector: name: pSB1C3_SDM_SspI '''P125'''
+
-
*Insert: name: pSB1C3_SDM_PvuII '''P129'''
+
-
*new vector name: pSB1C3_RFC25_SDM_SspI/PvuII '''P157'''
+
-
*buffer used: 4 ; Restriction-enzymes used: Enzyme 1 (no. Lab: 141) BtsI ; Enzyme 2 (no.Lab: 144) BpmI
+
-
*DNA concentration (vector): 321,9 ng/µl ; DNA concentration (insert): 308,4 µg/µl
+
-
 
+
-
<br />
+
{| border="1"
{| border="1"
-
| '''components'''   || align="right" |'''volume of pSB1C3_SDM_SspI /µl''' || align="right" |'''volume of pSB1C3_SDm_PvuII /µl'''  
+
| align="left" | '''Components''' ||align="left"| '''clone 1/µL''' ||align="left"| '''clone 2/µL''' ||align="left"| '''clone 3/µL'''||align="left"| '''original/µL'''
|-
|-
-
| DNA  || align="right" |4,66 || align="right" |4,87
+
| align="left" | DNA (500ng) ||align="left"| 1,4||align="left"|1,4||align="left"| 1,4||align="left"| 1,4
|-
|-
-
| BSA (10x) || align="right" |2 || align="right" | 2
+
| align="left" | BSA (10x) ||align="left"| 1||align="left"| 1||align="left"| 1||align="left"| 1
|-
|-
-
| Buffer 4 (10x)|| align="right" |2 || align="right" |2
+
| align="left" | Buffer 4 (10x) ||align="left"| 1||align="left"| 1||align="left"| 1||align="left"| 1
|-
|-
-
|Enzyme ClaI (no.Lab:152)|| align="right" |1 || align="right" |1
+
| align="left" | Enzyme 1 NgoMIV ||align="left"|0,5||align="left"| 0,5||align="left"|0,5||align="left"|0,5
|-
|-
-
|Enzyme BpmI (no.Lab:144)|| align="right" |1 || align="right" |1  
+
| align="left" | H<sub>2</sub>O ||align="left"|6,1||align="left"| 6,1||align="left"| 6,1||align="left"| 6,1
|-
|-
-
|H2O||  align="right" |9,34 || align="right" |9,13
+
| align="left" | '''Total volume'''  ||align="left"|10||align="left"| 10||align="left"| 10||align="left"| 10
-
|-
+
-
|'''Total volume (e.g. 15,20,25,30 µl)'''|| align="right" | 20|| align="right" |20
+
|}
|}
-
incubation time: 1,5h
 
<br />
<br />
-
0,5 g Agarose,50 ml TBE (1%), 3 µl GELRED (gel was shared with Anna) , at Volt, running time:
+
   
<br />
<br />
-
<br />
+
[[image:Freiburg10_Kopfkratzen.gif|thumb|right| Bitte schneid mich aus !]]
-
 
+
-
<br />
+
-
'''Loading plan for agarose gel''': <br>
+
-
Marker used: GeneRuler ladder mix (Fermentas)<br>
+
-
 
+
-
{| border="1"
+
-
|
+
-
!Marker
+
-
!Sample 125, 20µl
+
-
!Sample 129, 20µl
+
-
|-
+
-
!Lane
+
-
|1
+
-
|3
+
-
|5
+
-
|-
+
-
|}
+
-
This appproach also didn't work, same result. the guess is that BpmI doesn't work anymore. will be repeated by Chris W. on 05.08.10. the result will show that the guess is right and BpmI from labstock is changed out with a new one.
+
[[file:Freiburg10 test digestion NgoMIV.jpg]]
-
====<p style="font-size:15px; background-color:#66bbff;">'''Test transformation of XL1 competent cells'''</p>====
+
According to the gel, NgoMIV SDM was successful in clones 1 and 2. Clone 3 well reveales 2 bands of unknown origin. Samples 1 and 3 were sent for sequencing.
-
Investigator: Kira
+
-
Test transformation was performed in order to test the efficiency of produced chemical competent XL1 blue cells.
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Picking clones of pSB1C3_Affibody_linker and pSB1C3_ß-Globin_YFP_hGH_rITR  </b></p>====
 +
<b>Investigator: Anna </b>
-
2 plates were prepared: one with 50 pg and the second with 100 pg pUC 18 plasmid (50 pg/㎕)
+
<p style="font-size:13px; color:#003399;"><b>Comments</b>: A 1:1000 dilution of pSB1C3_Affibody_GSAT-Linker was prepared. <br/>
-
50 pg plate: 1 ㎕ pUC + 50 ㎕ XL1B cells
+
<b>To do</b>: Mini-Prep of pSB1C3_Affibody_(Short/Middle/Long and SEG-Linker) and pSB1C3_ß-Globin_YFP_hGH_rITR. Picking clones of pSB1C3_Affibody_GSAT-Linker. </p>
-
100 pg plate: 2 ㎕ pUC + 50 ㎕ XL1B cells
+
-
Transformation was performed according to the standard protocol and the plates were incubated at 37 C.
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Test digestion of pSB1C3_SDM_SspI_Bla14FM (P222), pSB1C3_BLA (P286, P288)  </b></p>====
 +
<b>Investigator: Achim</b>
 +
<p style="font-size:13px; color:#003399;"><b>Comments</b>New test digestion of two SDM-attempts, this time we also digested the original vector containing the PvuII restriction site to see differences.</p>
<br/>
<br/>
-
====<p style="font-size:15px; background-color:#ff00ff;">'''Sequencing results of ITRs'''</p>====
 
-
Investigator: <b>Hanna</b>
 
-
<br/>
 
-
8 sequencing files were analyzed "per hand" base by base. Alignments (will be inserted soon) delivered that the right ITR is 100% OK and was successfully converted into the RFC10 BioBrick standard. <br/>
 
-
[[File:Freiburg10_Sequencing_RFC10RightITR_2.jpg]] <br/>
+
[[File:Freiburg10 Test digestion of pSB1C3 BLA final 24 08 2010.jpg|800px]]
-
The sequencing and alignments of the left ITR delivered that a 15 bp fragment in the middle of the sequence is lacking. Therefore further test digestions have to be performed. Nevertheless also this ITR was successfully converted into the RFC10 BioBrick standard. Secondary structure analysis showed that the stemloop-structure will be nevertheless forming. We will try to test whether the referring fragment is also missing in the pAAV_MCS. If it's also lacking there we will continue with this ITR and test whether it functions.
 
-
<br/>
 
-
[[File:Freiburg10 Sequencing RFC10LeftITR 2.jpg]]
 
-
<br/>
 
-
<br/>
 
-
====<p style="font-size:15px; background-color:#66bbff;">'''Cloning of Rep68, Rep78 and AAP into pSB1C3'''</p>====
 
-
'''Investigator: Anna
 
-
<p style="font-size:13px; color:#68bbff;">'''''Comments''''': The PCR of Rep 40/52 didn't work (see 03.08), whereas the PCR of Rep 68/78 and AAP was succesful. Ligation was done with two different samples of pSB1C3 (see agarose gel). Samples from digestion and from ligation are stored in the 4°C freezer.</p> <br>
+
*The original vector is being cut as expected: the uncut vector can be seen in different conformations, the linearized vector shows one distinct band and the digestions cutting out BLA show a band at ~900 bp. Our SDM attempts show inconsistent results, not only does the Pvu site still seem to be in the backbone, there are no more proper insert bands visible either. We therefore conclude that either the DpnI digestion or the dilution of the transformation must have gone wrong (differing plasmids...). Tomorow we'll try a new cloning approach using an old vector with deleted Pvu restriction site and MscI & XbaI digestion. In case this also fails we also yet have to prep and test digest the repetition of the mutagenesis which was carried out by Stefan.
-
*Digestion of PCR products and vector:
 
 +
===100. labday 25.08.2010===
 +
====<p style="font-size:15px; background-color:#66bbFF;"><b>Cloning of pSB1C3_Rep40, pSB1C3_Rep68 and pMA_RC_insert </b></p>====
 +
'''Investigator: Chris L.'''<br>
 +
*buffer used: 2; Restriction-enzymes used: Enzyme 1: HindIII ; Enzyme 2: BstEII
 +
Plasmids:
 +
<ul><li>pSB1C3_Rep40 '''P231'''</li>
 +
<li>pSB1C3_Rep68 '''P266'''</li>
 +
Insert:
 +
<li>pMA_RC_insert '''P190'''</li>
 +
</ul>
<br />
<br />
{| border="1"
{| border="1"
-
| '''components'''   || align="right" |'''PCR product /µl''' || align="right" |'''vector /µl'''  
+
| align="left" | '''Components'''  ||align="left"| '''P190/µL''' ||align="left"| '''P231/µL''' ||align="left"| '''P266/µL'''  
|-
|-
-
| DNA  || align="right" |19 || align="right" |10
+
| align="left" | DNA  ||align="left"| 5.9 ||align="left"| 6.4 ||align="left"| 5
|-
|-
-
| BSA (10x) || align="right" |3 || align="right" | 3
+
| align="left" | BSA (10x) ||align="left"| 2 ||align="left"|2||align="left"| 2
|-
|-
-
| Buffer 4 (10x)|| align="right" |3 || align="right" |3
+
| align="left" | Buffer 2 (10x) ||align="left"| 2 ||align="left"| 2||align="left"| 2
|-
|-
-
|Enzyme '''XbaI || align="right" |1 || align="right" |1  
+
| align="left" | Enzyme 1 HindIII  ||align="left"| 1 ||align="left"| 1 ||align="left"| 1
|-
|-
-
|Enzyme '''SpeI || align="right" |1 || align="right" |1  
+
| align="left" | Enzyme 2 BstEII  ||align="left"| 1 ||align="left"| 1 ||align="left"| 1  
|-
|-
-
|H2O|| align="right" |3 || align="right" |12
+
| align="left" | H<sub>2</sub>O ||align="left"| 8.1 ||align="left"| 7.6 ||align="left"| 9
|-
|-
-
|'''Total volume (e.g. 15,20,25,30 µl)'''|| align="right" | 30|| align="right" |30
+
| align="left" | '''Total volume''' ||align="left"| 20 ||align="left"| 20||align="left"| 20
|}
|}
<br />
<br />
-
<p style="font-size:13px; color:#68bbff;">'''''Comments''''': Digestion was done with XbaI and SpeI, it has to be checked if the inserts are cloned into the vector in the right orientation. </p> <br>
+
*Incubation: 90 minutes; 37°C with HindIII<br>
-
 
+
*Incubation: 90 minutes; 60°C with BstEII<br>
-
*Purification of Rep68, 78 and AAP:
+
-
For the purification 95 µl of buffer PBI was used.
+
-
 
+
-
c(Rep68)= 17,6 ng/µl <br />
+
-
c(Rep78)= 80,60 ng/µl <br />
+
-
c(AAP)= 77,78 ng/µl
+
-
 
+
-
<br />
+
-
 
+
-
*Gelextraction of pSB1C3_RFC25_CFP:
+
-
0,5 g Agarose,50 ml TBE (1%), 3 µl GELRED , at 115 Volt, running time:55 <br />
+
-
5µl loading dye (6x) for the sample, Marker: GeneRuler ladder mix (Fermentas)
+
<br />
<br />
 +
'''Agarosegel'''
<br />
<br />
 +
0.5 g Agarose, 50 ml TAE (1 %), 3 µL GELRED, 5 min at 90 Volt, 40 min at 115 Volt
<br />
<br />
 +
[[File:Freiburg 10 pSB1C3 Rep40 pSB1C3 Rep68 pMA RC Insert.png|700px]]
<br />
<br />
 +
'''Concentrations''' measured via NanoDrop:
 +
*pSB1C3_Rep40: 81.97 ng/µl
 +
*pSB1C3_Rep68: 48,68 ng/µl
 +
*RC_Insert: 3,60 ng/µl
 +
<br>
 +
'''T4 Ligation of pSB1C3_Rep40 with RC_Insert'''<br>
 +
Volume insert: 6,54 µl<br>
 +
Volume vector: 1,46 µl<br>
 +
<br>
 +
'''T4 Ligation of pSB1C3_Rep68 with RC_Insert'''<br>
 +
Volume insert: 5,46 µl<br>
 +
Volume vector: 2,54 µl<br>
 +
<br>
 +
'''Trafo''' was prepared with XL1blue and Cm
-
[[file:Digestion of pSB1C3.jpg]]
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Picking clones of pAAV_RC-ins_rep_cap and pCeruleanVP1up</b></p>====
-
<br />
+
<b>Investigator: Chris L. </b>
 +
<b>To do</b>: Mini-Prep of pAAV_RC-ins_rep_cap and pCeruleanVP1up.
-
c(pSB1C3)= 2,87 ng/µl <br />
+
====<p style="font-size:15px; background-color:#66bbFF;"><b>Cloning of pSB1C3_6xHis with middlelinker and pSB1C3_6xHis with pGA14_mVenus_YFP</b></p>====
-
c(pSB1C3_2)= 9,46 ng/µl
+
'''Investigator: Jessica'''<br>
-
 
+
*buffer used: 4; Restriction-enzymes used: Enzyme 1: AgeI ; Enzyme 2: PstI ; Enzyme3: NgoMIV
-
<br />
+
<ul><li>Plasmids:</li>
-
 
+
<ul><li>pSB1C3_6xHis '''P84'''</li>
-
*Quickligation of PCR products and vector:
+
<li>pGA14_middle linker '''P65'''</li>
-
 
+
<li>GA14_mVenus_YFP '''P60'''</li>
-
For the Ligation 10µl buffer (2x) and 1µl Quickligase were used.
+
-
<br />
+
-
{| border="1"
+
-
| ''' '''  || align="right" |'''Sample-no.'''|| align="right" |'''vector /µl''' || align="right" |'''insert /µl'''
+
-
|-
+
-
| pSB1C3 + Rep68 ||  align="right" |1.1 || align="right" |6,51 ||  align="right" |2,4
+
-
|-
+
-
| pSB1C3_2 + Rep68 ||  align="right" |1.2 ||  align="right" |3,9 ||  align="right" | 5,02
+
-
|-
+
-
| pSB1C32 + Rep78 ||  align="right" |2.1 || align="right" |8,2 ||  align="right" |0,8
+
-
|-
+
-
| pSB1C3_2 + Rep78 ||  align="right" |2.2 ||  align="right" |6,82 ||  align="right" |2,18
+
-
|-
+
-
| pSB1C3 + AAP ||  align="right" |3.1 ||  align="right" |8,71 ||  align="right" |0,92
+
-
|-
+
-
| pSB1C3_2 + AAP  ||  align="right" |3.2 ||  align="right" |8,1 ||  align="right" |0,9
+
-
|-
+
-
|'''Total volume (e.g. 15,20,25,30 µl)'''||  align="right" | 9||  align="right" | ||  align="right" |
+
-
|}
+
-
<br />
+
-
 
+
-
*Transformation:
+
-
 
+
-
The transformation was done following the standard protocol using B21 cells.
+
-
 
+
-
===80.Labortag 05.08.2010===
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>New LB Agar was prepared.</b></p>====
+
-
Investigator: Patrick
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Sequenc analysis of BioBricks: CMV, hGH and beta globin</b></p>====
+
-
<b>Investigator: Bea</b><br />
+
-
<p style="font-size:13px; color:#68bbff;"><b>Comments</b>: Sequence analysis of three BioBricks which were cloned into the iGEM standard plasmid pSB1C3. Cloning of this three plasmids were performed at: </p> <br/ >
+
-
<ul>
+
-
<li> </li>
+
-
<li>   </li>
+
-
<li>   </li>
+
</ul>
</ul>
-
<p style="font-size:13px; color:#b2222;"><b>Sequencing results of pSB1C3_CMV: </b></p>
 
-
<br />
 
-
<ul>
 
-
<li>Plasmid sent for sequencing: </li>
 
-
<li>Primer used: VR-2 </li>
 
-
<li><b>Results:</b> Sequence read looks good. The incorporation of the PCR product can be confirmed. It can be seen that the insert is in the RFC10 standard and prefix and suffix have the right sequence. Therefore, this BioBrick can be send to the registry and used for BioBrick assembly.But: there is a mutation in the termintor (sequence picture do not show this mutation)</li>
 
</ul>
</ul>
-
<img src="http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/7/74/Freiburg10_Sequence_analysis_of_PSB1C3_CMV.jpg" />
 
<br />
<br />
-
<p style="font-size:13px; color:#b2222;"><b>Sequencing results of pSB1C3_hGH: </b></p>
 
-
<br />
 
-
<ul>
 
-
<li>Plasmid sent for sequencing: </li>
 
-
<li>Primer used: VR-2 </li>
 
-
<li><b>Results:</b> Sequence read looks good. The incorporation of the PCR product can be confirmed. It can be seen that the insert is in the RFC10 standard and prefix and suffix have the right sequence. Therefore, this BioBrick can be send to the registry and used for BioBrick assembly.</li>
 
-
</ul>
 
-
http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/e/e1/Freiburg10_Sequence_analysis_of_PSB1C3_betaglobin.jpg
 
-
<br />
 
-
<p style="font-size:13px; color:#b2222;"><b>Sequencing results of pSB1C3_hGH: </b></p>
 
-
<br />
 
-
<ul>
 
-
<li>Plasmid sent for sequencing: </li>
 
-
<li>Primer used: VR-2 </li>
 
-
<li><b>Results:</b> Sequence read looks good. The incorporation of the PCR product can be confirmed. It can be seen that the insert is in the RFC10 standard and prefix and suffix have the right sequence. Therefore, this BioBrick can be send to the registry and used for BioBrick assembly.</li>
 
-
</ul>
 
-
http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/4/44/Freiburg10_Sequence_analysis_of_PSB1C3_hGH.jpg
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b> Cellculture </b></p>====
 
-
Investigator: Adrian<br />
 
-
<br />
 
-
<br />
 
-
The Motivation: Transfection<br />
 
-
The Action: HEK cells were split into four T75 flasks<br />
 
-
The Plan: The cells should be ready at saturday for seeding and monday for transfection<br />
 
-
<br /><br />
 
-
We recived new HT1080 cells, they'll be split tomorrow (thx to Sven)
 
-
<br /><br />
 
-
We recived new tumor cells which overexpress EGFR (thx to barbara)
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Picking clones of pSB1C3_AAP_2, pSB1C3_AAP, pSB1C3_Rep78_2, pSB1C3_Rep78, pSB1C3_Rep68_2 and pSB1C3_Rep68</b></p>====
 
-
<br/>
 
-
Investigator: Anissa
 
-
<br/>
 
-
Of each construct two clones were picked. plates are still stored in the cold-room, tomorrow mini-prep will be done.
 
-
<br />
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>PCR and ligation for biobrick-production of pSB1C3_hTERT</b></p>====
 
-
<br/>
 
-
Investigator: Anissa,Kerstin<br />
 
-
<br />
 
-
<br />
 
-
<p style="font-size:13px; color:#68bbff;">'''''Comments''''': PCR was performed one time without DMSO and one time with DMSO. Only the approach with DMSO showed bands in the analytic gel after PCR. That's why only this approach will be noted. </p> <br>
 
-
<br>
 
-
*'''PCR:
 
-
(was performed following the standard protocol)
 
-
<br>
 
{| border="1"
{| border="1"
-
| '''Ingredients''' || align="right" |'''Volume / µl'''  
+
| align="left" | '''Components'''  ||align="left"| '''Mastermix'''  ||align="left"| '''P84/µL''' ||align="left"| '''P65/µL''' ||align="left"| '''P60/µL'''
|-
|-
-
| 5X Phusion HF buffer ||  align="right" |10
+
| align="left" | DNA ||align="left"| -||align="left"| 13,91||align="left"| 13,52||align="left"| 8,93
|-
|-
-
| 10 mM dNTP mix|| align="right" |1
+
| align="left" | BSA (10x) ||align="left"|-||align="left"|2||align="left"| 2||align="left"| 2
|-
|-
-
| forward primer: O111 (phTERT_prefix_for_RFC10) || align="right" |2,5
+
| align="left" | Buffer 4 (10x) ||align="left"| -||align="left"| 2||align="left"| 2||align="left"| 2
|-
|-
-
| reverse primer: O112 (phTERT_suffix_rev_RFC10) || align="right" |2,5
+
| align="left" | Enzyme 1 AgeI  ||align="left"|-||align="left"| 1||align="left"| -||align="left"| -
|-
|-
-
| DNA Template||  align="right" |0,35 µl
+
| align="left" | Enzyme 2 PstI  ||align="left"| -||align="left"| 1||align="left"| 1 ||align="left"| 1
|-
|-
-
| DMSO (2%)||  align="right" | 1
+
| align="left" | Enzyme 3 NgoMIV  ||align="left"| -||align="left"| -||align="left"| 1||align="left"| 1
|-
|-
-
| Phusion Polymerase||  align="right" |0,5
+
| align="left" | H<sub>2</sub>O ||align="left"| -||align="left"| 0,09||align="left"| 0,48||align="left"| 5,07
-
|-
+
-
|H<sub>2</sub>O|| align="right" |32,15
+
|-
|-
-
|Total volume|| align="right" |50
+
| align="left" | '''Total volume'''  ||align="left"| -||align="left"| 20||align="left"| 20||align="left"| 20
|}
|}
-
<br>
 
-
 
-
PCR program:
 
-
{| border="1"
 
-
|'''PCR Program'''|| align="right" |'''temperature/ °C'''
 
-
|-
 
-
|1||  align="right" |98                               
 
-
|-
 
-
|2||  align="right" |98
 
-
|-
 
-
|8x||  align="right" |58 
 
-
|-
 
-
|3 ||  align="right" |72
 
-
|-
 
-
|4 ||  align="right" |98
 
-
|-
 
-
|17x||  align="right" |70
 
-
|-
 
-
|5||  align="right" |72
 
-
|-
 
-
|6x||  align="right" |72
 
-
|-
 
-
|Hold||  align="right" |4
 
-
|}
 
-
<br>
 
-
 
-
 
-
<gallery widths=500px heights=300px perrow=2 caption="BioBrick production of pSB1C3_hTERT">
 
-
Image:Freiburg10_pSB1C3 cut with XbaI and PstI for TERT promoter.jpg|Preparative gel
 
-
Image:Freiburg10_hTERT after PCR.jpg|analytic gel
 
-
</gallery>
 
-
<br/>
 
-
 
-
<p style="font-size:15px; font-weight: bold; color: blue;">Gel extraction</p>
 
<br />
<br />
-
Gel measurement:
 
-
<br />
 
-
{| border="1"
 
-
| align="left" | '''Sample'''
 
-
| align="left" | '''Weight'''
 
-
| align="left" | '''Volume'''
 
-
| align="left" | '''Concentration'''
 
-
|-
 
-
| align="left" | hTERT
 
-
| align="left" | 0,15g
 
-
| align="left" | 20 µl
 
-
| align="left" | 37,49 ng/µl
 
-
|-
 
-
| align="left" | pSB1C3_cut
 
-
| align="left" | 0,08g
 
-
| align="left" | 20 µl
 
-
| align="left" | 4,56 ng/µl
 
-
|}
 
-
<br>
 
-
 
-
*Digestion of PCR product and vector:
 
 +
*Incubation:110 minutes; 37°C<br>
 +
'''Agarosegel'''
<br />
<br />
-
{| border="1"
+
0.5 g Agarose, 50 ml TAE (1 %), 3 µL GELRED, at 120 Volt
-
| '''components'''  || align="right" |'''PCR product /µl''' || align="right" |'''vector /µl'''
+
-
|-
+
-
| DNA  ||  align="right" |18||  align="right" |7,58
+
-
|-
+
-
| BSA (10x) ||  align="right" |0,3 (100X used)||  align="right" | 2
+
-
|-
+
-
| Buffer 4 (10x)||  align="right" |2,5 ||  align="right" |2
+
-
|-
+
-
|Enzyme '''XbaI ||  align="right" |1 ||  align="right" |1
+
-
|-
+
-
|Enzyme '''PstI ||  align="right" |1 ||  align="right" |1
+
-
|-
+
-
|H2O||  align="right" |2,2 ||  align="right" |6,42
+
-
|-
+
-
|'''Total volume (e.g. 15,20,25,30 µl)'''||  align="right" | 25||  align="right" |20
+
-
|}
+
<br />
<br />
-
Ligation with T4-Ligase and transformation with BL 21 was made. Clones have to be picked tomorrow.
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Transformation evaluation of XL1B cells and repetition of transformation</b></p>====
 
-
 
-
Investigator: Kira <br />
 
-
Against our expectations both agar plates contain very few colonies. In order to figure out if the lack of colonies is due to the XL1 blue cells or loss of pUC activity, test transformation will be repeated this evening with recently prepared XL1 blue cells as well as with Lab-XL1B cells.
 
-
50 pg pUC-plate contains 8 colonies
 
-
100 pg pUC-plate contains 18 colonies
 
-
Transformation will be performed again with 100 pg pUC (= 2 ㎕ pUC).
 
<br />
<br />
-
====<p style="font-size:15px; background-color:#66bbff;">'''Cloning of SDM SspI and SDM PvuII'''</p>====
 
-
'''Investigator: Chris W.'''
 
-
*Vector: name: pSB1C3_SDM_SspI '''P125'''
 
-
*Insert: name: pSB1C3_SDM_PvuII '''P129'''
 
-
*new vector name: pSB1c3_SDM_SspI/PvuII '''P157'''
 
-
*buffer used: 4 ; Restriction-enzymes used: Enzyme 1 (no. Lab: 144) BpmI ; Enzyme 2 (no.Lab: 152) ClaI
 
-
*DNA concentration (vector): 321,9 ng/µl ; DNA concentration (insert): 308,4 µg/µl
 
<br />
<br />
-
{| border="1"
+
[[File:Freiburg10 gel cut n terminus1.jpg|500px|left|thumb|]]
-
| '''components'''  || align="right" |'''volume of pSB1C3_SDM_SspI /µl''' || align="right" |'''volume of pSB1C3_SDm_PvuII /µl'''
+
-
|-
+
-
| DNA  ||  align="right" |4,66 ||  align="right" |4,87
+
-
|-
+
-
| BSA (10x) ||  align="right" |2 ||  align="right" | 2
+
-
|-
+
-
| Buffer 4 (10x)||  align="right" |2 ||  align="right" |2
+
-
|-
+
-
|Enzyme BpmI (no.Lab:144)||  align="right" |1 ||  align="right" |1
+
-
|-
+
-
|Enzyme ClaI (no.Lab:152)||  align="right" |1 ||  align="right" |1
+
-
|-
+
-
|H2O||  align="right" |9,34 ||  align="right" |9,13
+
-
|-
+
-
|'''Total volume (e.g. 15,20,25,30 µl)'''||  align="right" | 20||  align="right" |20
+
-
|}
+
-
<br> <p style="color:red;">Comment: I guess enzyme 144 was BpmI and enzyme 152 was ClaI ? (Jessica)</p> <br>
+
-
<br> <p style="color:red;">Comment: u r right, changed (Chris)</p> <br>
+
-
<br />
+
-
0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 130 Volt, running time:45
+
-
<br />
+
-
<br />
+
-
 
+
-
<br />
+
-
'''Loading plan for agarose gel''': <br>
+
-
Marker used: GeneRuler ladder mix (Fermentas)<br>
+
-
 
+
-
{| border="1"
+
-
|
+
-
!Marker
+
-
!Sample 125, 20µl
+
-
!Sample 129, 20µl
+
-
|-
+
-
!Lane
+
-
|1
+
-
|5
+
-
|7
+
-
|-
+
-
|}
+
<br>
<br>
-
[[File:Gel1.png|500px|left|thumb|]]
 
<br>
<br>
<br>
<br>
Line 1,545: Line 725:
<br>
<br>
<br>
<br>
-
 
+
'''concentrations''' measured via NanoDrop:
-
 
+
*SB1C3_6xHis: 1,7 ng/µl
-
The mutual vector (now without SspI) and insert (now without PvuII) were cut out. The gelextraction was performed according to the standard protocol. DNA concentration of the extracts:
+
*middle linker: 3,8 ng/µl
-
* P125 Sspl: 12,8 ng/µl
+
*mVenus_YFP: 3,2 ng/µl
-
* P129 PvuII: 11,6 ng/µl
+
<br>
 +
'''T4 Ligation pSB1C3_6xHis_middlelinker'''<br>
 +
Volume insert: 0,25 µl<br>
 +
Volume vector: 7,75 µl<br>
<br>
<br>
-
The  Ligation was performed as following:
+
'''T4 Ligation pSB1C3_6xHis_mVenus_YFP'''<br>
-
* Vector Volume: 2,81 µl
+
Volume insert: 2,64 µl<br>
-
* Insert Volume: 5,19 µl
+
Volume vector: 5,36 µl<br>
<br>
<br>
-
* 1µl T4-Ligase buffer (10x)
+
'''Trafo''' was prepared with XL1blue and Cm
-
* 8µl (Vector + Insert) mix
+
-
* 1µl T4-Ligase
+
-
<br> Incubate for 30min
+
 +
====<p style="font-size:15px; background-color:#66bbff;"><b>Sequence analysis of pSB1C3_6xHis</b></p>====
 +
'''Investigator: Jessica'''<br>
 +
'''P84''' was sequenced for continuation of working with it
 +
<br>
<br>
<br>
-
'''Transformation:''' performed according to the standard protocol (BL21). The cells were plated on a agar plate with chloramphenicol
 
<br>
<br>
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Miniprep of p158 and p159</b></p>====
+
[[File:Freiburg10 pSB1C3_6xHis.jpg|900px]]
 +
<br>
 +
* '''pSB1C3_6xHis P84 is ready to use, but is empty. a new tube will be made tomorrow'''
-
<p style="font-size:20px; font-weight: bold; color: blue;"><u>Plasmid Mini-Prep</u></p>
+
====<p style="font-size:15px; background-color:#66bbFF;"><b>Inoculation of pGA14_middlelinker and pSB1C3_6xHis clone1 </b></p>====
-
Investigator: Bea & Volker<br>
+
'''Investigator: Jessica'''<br>
-
For the tripple mutant of pAAV-RC a Quick-change reaction was carried out to remove the last restriction site (SalI) that is required for the Viral Brick standard. For this construct glycerol stocks and minipreps were carried out for two clones.<br>
+
*both tubes ('''P65''' and '''P84''')are empty --> inoculation with glycerolstocks
 +
**'''B48''' pGA14_middlelinker was inoculated with 10ml DYT and 10µl Amp
 +
**'''B64''' pSB1C3_6xHis clone1 was inoculated with 10ml DYT and 10µl Cm
-
<br />
+
====<p style="font-size:15px; background-color:#66bbff;">'''Cloning of pSB1C3_SspI_BLA and pSB1C3_CFP_PvuII'''</p>====
-
<u>Glycerol Stocks</u>
+
-
{| border="1"
+
-
| align="left" |  ||align="left"| '''Clone 1''' ||align="left"| '''Clone 2'''
+
-
|-
+
-
| align="left" | '''Bacteria strain''' ||align="left"| BL-21 ||align="left"| BL-21
+
-
|-
+
-
| align="left" | '''Plasmidname''' ||align="left"| pAAV_RC_1.2 SDM SalI
+
-
||align="left"| pAAV_RC_1.2 SDM SalI
+
-
|-
+
-
| align="left" | '''Date''' ||align="left"| 05.08.2010 ||align="left"| 05.08.2010
+
-
|-
+
-
| align="left" | '''given glycerol-stock no.''' ||align="left"| B126 ||align="left"| B127
+
-
|-
+
-
| align="left" | '''given plasmid no.''' ||align="left"| p158 ||align="left"| p159
+
-
|}
+
-
<br />
+
-
TO do: Test digestion and sequencing!!!
+
-
===81.Labortag 06.08.2010===
+
<b> Investigator: Achim</b>
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Cloning Rep40 & Rep52 into pSB1C3</b></p>====
+
-
Intention: get biobrick ready <br>
+
-
Investigator: Patrick <br>
+
-
PCR of pKEX-2XL.Rep 40 (P22) and pKEX-2XL.Rep 52 (P23) following gelrun (1%) and gelextraction. <br>
+
-
Used Primers:
+
-
* Praefix Rep40_52ex (O94)
+
-
* Suffix Rep 40_68ex (O96)
+
-
* Suffix Rep 52_78ex (O97)
+
-
<br>
+
-
PCR programm of Rep40:                         
+
-
*98°C  1 min
+
-
<br>
+
-
*98°C  15 sec
+
-
*63°C  25 sec
+
-
*72°C  15 sec  Repeat this cycle 8 times.
+
-
<br>
+
-
*98°C  15 sec
+
-
*68°C  25 sec
+
-
*72°C  15 sec  Repeat this cycle 17 times.
+
-
<br>
+
-
*72°C  5 min
+
-
*Hold 4°C
+
-
<br>
+
-
<br>
+
-
PCR programm of Rep52:                         
+
-
*98°C  1 min
+
-
<br>
+
-
*98°C  15 sec
+
-
*62°C  25 sec
+
-
*72°C  20 sec  Repeat this cycle 8 times.
+
-
<br>
+
-
*98°C  15 sec
+
-
*66°C  25 sec
+
-
*72°C  20 sec  Repeat this cycle 17 times.
+
-
<br>
+
-
*72°C  5 min
+
-
*Hold 4°C
+
-
<br>
+
<p style="font-size:13px; color:#003399;"><b>Comment</b>:I ligated a sequence from pSB1C3_CFP_PvuII missing the PvuII restriction site into pSB1C3_SspI_BLA to obtain a pSB1C3 vector without PvuII in the cat marker.</p>
-
{| border="1"
+
-
|'''ingredients'''|| align="right" |'''Rep 40'''|| align="right" |'''Rep40 + DMSO'''|| align="right" |'''Rep52'''|| align="right" |''' Rep52 + DMSO
+
-
|-
+
-
| 5x Phusion HF buffer||  align="right" |10 µl || align="right" |10 µl|| align="right" |10 µl|| align="right" |10 µl                             
+
-
|-
+
-
|10 mM dNTP mix||  align="right" |1 µl || align="right" |1 µl|| align="right" |1 µl|| align="right" |1 µl
+
-
|-
+
-
|for Primer O94 (1:10 dilution, 05 µM)||  align="right" |2,5 µl  || align="right" |2,5 µl|| align="right" |2,5 µl|| align="right" |2,5 µl
+
-
|-
+
-
|rev Primer (1:10 dilution, 0,5 µM) ||  align="right" |2,5 µl O96|| align="right" |2,5 µl O96|| align="right" |2,5 µl O97|| align="right" |2,5 µl O97
+
-
|-
+
-
|DNA template ||  align="right" | 1 µl, 112,7 ng/µl|| align="right" |1 µl, 112,7 ng/µl|| align="right" |0,9 µl, 142,3 ng/µl|| align="right" |0,9 µl, 142,3 ng/µl
+
-
|-
+
-
|DMSO||  align="right" |0 µl|| align="right" |0,5 µl|| align="right" |0 µl|| align="right" |0,5 µl
+
-
|-
+
-
|Phusion Polymerase||  align="right" |0,5 µl|| align="right" |0,5 µl|| align="right" |0,5 µl|| align="right" |0,5 µl
+
-
|-
+
-
|H2O||  align="right" |32,5 µl|| align="right" |32 µl|| align="right" |32,6 µl|| align="right" |32,1 µl
+
-
|-
+
-
|Total volume||  align="right" |50 µl|| align="right" |50 µl|| align="right" |50 µl|| align="right" |50 µl
+
-
|}
+
-
<br>
+
-
Ecpected size of Rep40: 940 bp, expected size of Rep52:1198 bp. There are two samples of Rep40 and Rep52 and the PCR of one of each was run with 1% DMSO because the Rep40 Praefix has a strong secondary structure and was used as a primer for Rep 40 and Rep 52<br>
+
Vector: <br>
-
<br>
+
*pSB1C3_SspI_BLA (p222): c= 308,32 ng/µl<br>
-
[[File:IM000026_Patrick.JPG|400px]]
+
Insert: <br>
-
<br>
+
*pSB1C3_CFP_PvuII (p129): c= 264,86 ng/µl<br>
-
<br>
+
-
Digestion of pSB1C3 (P51.1) with EcoRI-HF and SpeI following gelrun (0.8%) and gelextraction. <br>
+
-
Expected size of the fragments: about 2000 bp and 800bp <br>
+
-
<br>
+
-
[[File:IM000028_Patrick.JPG‎|500px]]
+
-
<br>
+
-
<br>
+
-
All marked fragments were extracted. The Gelextraction was performed according to the standard protocol following a digestion of the PCR products with EcoRI HF and SpeI and a purification of the PCR product. The Ligation was not performed according to the standard protocol: 1 µl T4 DNA Ligase, 1 µl (10x) Buffer, 3 µl vector pSB1C3 (60 ng/µl) and 5 µl insert (all concentrations about 100 ng/µl). The Transformation (with BL21) was performed according to the standard protocol. Tomorrow there will hopefully be some clones on the agar plates.
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>BioBrick Assembly of pSB1C3_beta globin_mVenus</b></p>====
 
-
Investigator: <b>Bea </b>
 
-
<br />
 
-
<p style="font-size:13px; color:#003399;"><b>Comments</b>: BioBricks are ready to use. The sequenced pSB1C3_beta globin (P133) and pGA14_mVenus (P60) will be digested with different enzymes and will be assembled in order to produce the first step in assembling the whole vector together. </p>
 
-
<br />
 
-
<ul>
 
-
<li>First the plasmids P133 and P60 were digested:</li>
 
-
</ul>
 
-
<table border=2 cellpadding=1 cellspacing=1 width=528 style='border-collapse:
 
-
collapse;table-layout:fixed;width:396pt'>
 
-
<tr height=20 style='height:15.0pt'>
 
-
  <td height=20 class=xl65 width=176 style='height:15.0pt;width:132pt'>&nbsp;</td>
 
-
  <td class=xl66 width=176 style='border-left:none;width:132pt'>pSB1C3_betaglobin/µL</td>
 
-
  <td class=xl66 width=176 style='border-left:none;width:132pt'>pGA14_mVenus/µL</td>
 
-
</tr>
 
-
<tr height=20 style='height:15.0pt'>
 
-
  <td height=20 class=xl67 width=176 style='height:15.0pt;border-top:none;
 
-
  width:132pt'>DNA</td>
 
-
  <td class=xl68 style='border-top:none;border-left:none'>7</td>
 
-
  <td class=xl68 style='border-top:none;border-left:none'>9</td>
 
-
</tr>
 
-
<tr height=20 style='height:15.0pt'>
 
-
  <td height=20 class=xl67 width=176 style='height:15.0pt;border-top:none;
 
-
  width:132pt'>BSA (100x)</td>
 
-
  <td class=xl68 style='border-top:none;border-left:none'>0,2</td>
 
-
  <td class=xl68 style='border-top:none;border-left:none'>0,2</td>
 
-
</tr>
 
-
<tr height=40 style='height:30.0pt'>
 
-
  <td height=40 class=xl67 width=176 style='height:30.0pt;border-top:none;
 
-
  width:132pt'>Buffer 4<span style='mso-spacerun:yes'>  </span>(10x)</td>
 
-
  <td class=xl68 style='border-top:none;border-left:none'>2</td>
 
-
  <td class=xl68 style='border-top:none;border-left:none'>2</td>
 
-
</tr>
 
-
<tr height=40 style='height:30.0pt'>
 
-
  <td height=40 class=xl67 width=176 style='height:30.0pt;border-top:none;
 
-
  width:132pt'>Enzyme XbaI<span style='mso-spacerun:yes'> </span></td>
 
-
  <td class=xl68 style='border-top:none;border-left:none'>1</td>
 
-
  <td class=xl68 style='border-top:none;border-left:none'>1</td>
 
-
</tr>
 
-
<tr height=40 style='height:30.0pt'>
 
-
  <td height=40 class=xl67 width=176 style='height:30.0pt;border-top:none;
 
-
  width:132pt'>Enzyme AgeI</td>
 
-
  <td class=xl68 style='border-top:none;border-left:none'>1</td>
 
-
  <td class=xl68 style='border-top:none;border-left:none'>1</td>
 
-
</tr>
 
-
<tr height=20 style='height:15.0pt'>
 
-
  <td height=20 class=xl67 width=176 style='height:15.0pt;border-top:none;
 
-
  width:132pt'>H2O</td>
 
-
  <td class=xl68 style='border-top:none;border-left:none'>8,8</td>
 
-
  <td class=xl68 style='border-top:none;border-left:none'>6,8</td>
 
-
</tr>
 
-
<tr height=40 style='height:30.0pt'>
 
-
  <td height=40 class=xl67 width=176 style='height:30.0pt;border-top:none;
 
-
  width:132pt'>Total volume</td>
 
-
  <td class=xl69 style='border-top:none;border-left:none'>20</td>
 
-
  <td class=xl69 style='border-top:none;border-left:none'>20</td>
 
-
</tr>
 
-
<tr height=0 style='display:none'>
 
-
  <td width=176 style='width:132pt'></td>
 
-
  <td width=176 style='width:132pt'></td>
 
-
  <td width=176 style='width:132pt'></td>
 
-
</tr>
 
-
</table>
 
-
<ul>
 
-
<li> Incubation of plasmids at 37°C for 2 hours </li>
 
-
<li> Load samples on preparative 1% agarose gel and run at 110 V, 45 minutes </li>
 
-
</ul>
 
-
<br/>
 
-
<b>Results:</b> As it can be seen in the picture below, digestion of the vector pSB1C3_betaglobin (left lane) revealed a band at around 2500 - 3000 bp. The expected size after digetsing with SpeI and PstI was: 2555 bp. The band ran a little bit higher than expected, anyhow the band was cut out of the gel. The right lane belongs to the pGA14_mVenus which was digested with XbaI and PstI which resulted in two fragments. mVENUS was digested. Expected sizes were: mVenus = 756bp and pGA14 = 2900bp. The expected sizes correspond to the bands seen in the picture below. The smaller fragment belonging to mVenus was cut out of the gel as well.
 
-
<br />
 
-
<br />
 
-
[[File:Freiburg10 pSB1C3 betaglobin mVenus 06 08 2010.jpg|550px]]
 
-
<br />
 
-
<br />
 
-
After gel extraction has been performed, the concentrations of the two fragments were measured and we obtained following concentrations:
 
-
<ul>
 
-
<li>c(pSB1C3_betaglobin) = 15,62 ng/µL </li>
 
-
<li>c(mVenus) = 6,83  ng/µL </li>
 
-
</ul>
 
-
<br />
 
-
For ligation the T4-Ligase were used.
 
-
<ul>
 
-
<li>v(pSB1C3_betaglobin) = 2,64 µL </li>
 
-
<li>v(mVenus) =5,36 µL </li>
 
-
<li>v(10xT4 Ligase buffer) = 1 µL </li>
 
-
<li>v(T4-Ligase) = 1 µL </li>
 
-
<li><b>Total volume = 10 µL </b></li>
 
-
</ul>
 
-
<br />
 
-
Additionally a control ligation was performed.
 
-
<ul>
 
-
<li>v(pSB1C3_betaglobin) = 2,64 µL </li>
 
-
<li>v(H<sub>2</sub>0) =5,36 µL </li>
 
-
<li>v(10xT4 Ligase buffer) = 1 µL </li>
 
-
<li>v(T4-Ligase) = 1 µL </li>
 
-
<li><b>Total volume = 10 µL </b></li>
 
-
<li>Ligation duration was 45 minutes on room temperature.</li>
 
-
</ul>
 
-
<br />
 
-
After ligation a transformation was performed.
 
-
<ul>
 
-
<li> Used bacterial strain: XL1-B</li>
 
-
<li> LB-agar plates containing chloramphenicol were plated and put in the 37°C room over night</li>
 
-
</ul>
 
-
<br />
 
-
<p style="font-size:13px; color:#003399;"><b>To do</b>: Mini-Prep and test digestion in order to verify assembly of beta globin and YFP. After verification of the fusion of the two fragments this construct is ready for the next BioBrick assembly step. </p>
 
-
====<p style="font-size:15px; background-color:#ff00ff;">'''Test digestion of SalI SDM'''</p>====
 
-
Investigator: <b>Hanna</b>
 
-
<br/>
 
-
<p style="font-size:15px; font-weight: bold; color:#ff00ff;">Test digestion</p>
 
-
<ul>
 
-
<li>buffer used: 3 ; Restriction-enzymes used: Enzyme 1 (no. Lab: 53): SalI ; Enzyme 2: XbaI </li>
 
-
<li>Plasmid
 
-
<ul>
 
-
<li>Given Plasmid-Number: P158; DNA concentration: 398.8 ng/µL ;</li>
 
-
<li>Given Plasmid-Number: P159; DNA concentration: 437.4 ng/µL ;</li>
 
-
</ul>
 
-
</ul>
 
-
<br />
 
-
<br />
 
{| border="1"
{| border="1"
-
| align="left" | '''Components''' ||align="left"| '''P158 Volume/µL''' ||align="left"| '''P159 Volume/µL'''
+
| components  || align="right" |Vector || align="right" |Insert
|-
|-
-
| align="left" | DNA ||align="left"| 2.5 ||align="left"| 2.3
+
| DNA || align="right" |3,8 || align="right" | 4,9
|-
|-
-
| align="left" | BSA (10x) ||align="left"| 1 ||align="left"| 1
+
| BSA (10x) || align="right" |2|| align="right" | 2
|-
|-
-
| align="left" | Buffer 3 (10x) ||align="left"| 1 ||align="left"| 1
+
| Buffer 4 (10x)|| align="right" |2 || align="right" | 2
|-
|-
-
| align="left" | SalI (no. Lab: 53) ||align="left"| 0.5 ||align="left"| 0.5
+
|Enzyme MscI || align="right" |1 || align="right" | 1
|-
|-
-
| align="left" | XbaI ||align="left"| 0.75 ||align="left"| 0.75
+
|Enzyme XbaI || align="right" |1|| align="right" | 1
|-
|-
-
| align="left" | H<sub>2</sub>O ||align="left"| 4.25 ||align="left"| 4.45
+
|H<sub>2</sub>O|| align="right" |10,2 || align="right" | 9,1
|-
|-
-
| align="left" | <b>Total volume</b> ||align="left"| <b>10</b> ||align="left"| <b>10</b>
+
|'''Total volume '''|| align="right" |20 || align="right" | 20
|}
|}
 +
<br>
 +
 +
*Gelextraction:
 +
0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 110 Volt, running time:45 <br />
 +
Marker: GeneRuler ladder mix (Fermentas)
<br />
<br />
-
Incubation: 1 h
 
<br />
<br />
-
<p style="font-size:15px; font-weight: bold; color:#ff00ff;">Agarose-Gel:</p>
 
<br />
<br />
-
0.5  g Agarose, 50 mL TBE (1 %),3 µL GELRED, at 115 Volt, running time: 45 minutes
 
<br />
<br />
 +
 +
[[Image:Freiburg10 25082010achim.JPG|400px]]
<br />
<br />
-
{| border="1" cellspacing="0" cellpadding="2" bordercolor="black"
+
 
-
!Sample
+
 
-
!Sample/µl]
+
 
-
!Loading dye (5x)/µl
+
*c(Insert)= 15,66 ng/µl; size: 730 bp<br />
-
!Expected size 1 (if SDM didn't work)
+
*c(Vector)= 14,42 ng/µl; size: 2200 bp
-
!Expected size 2 (if SDM didn't work)
+
 
-
!Expected size 3 (if SDM didn't work)
+
-
|--
+
-
|P158
+
-
|10 µl
+
-
|2 µl
+
-
|6129 bp
+
-
|1143 bp
+
-
|61 bp
+
-
|--
+
-
|P159
+
-
|10 µl
+
-
|2 µl
+
-
|6129 bp
+
-
|1143 bp
+
-
|61 bp
+
-
|--
+
-
|}
+
-
{| align=right
+
-
|}
+
<br />
<br />
-
*Marker: GeneRuler ladder mix
 
-
{| border="1"
 
-
|
 
-
!Marker /µL
 
-
!Sample P158 /µl
 
-
!Sample P159 /µl
 
-
!Marker /µL
 
-
|-
 
-
!Lane
 
-
|6.5
 
-
|10
 
-
|10
 
-
|7
 
-
|-
 
-
|}
 
-
<br />
 
-
[[File:Freiburg10 Test digestion SalI1.jpg|600px]] <br/>
 
-
<b>Comments:</b> Test digestion looked good: No 1143 bp fragment was detectable in neither test digestion. <br/>
+
*Ligation of PCR products and vector:
-
P158 was sent for sequencing to GATC. <br/>
+
-
====<p style="font-size:15px; background-color:#ff00ff;">'''ITR test digestion of pAAV_MCS'''</p>====
+
For the Ligation 1µl T4 buffer (2x) and 1µl T4 ligase were used. Incubation time: 60 min due to blunt end ligation
-
Investigator: <b>Hanna</b>
+
-
<br/>
+
-
<p style="color:#ff00ff;"><b>Comment:</b> Because sequencing of the right ITR delivered that perhaps a 15 bp fragment is missing in the sequence, we wanted to check, whether this fragment is also not present in the original pAAV_MCS plasmid or whether the loss of these 15 bp happened during cloning. </p>
+
-
<br/>
+
-
<p style="font-size:15px; font-weight: bold; color:#ff00ff;">Test digestion</p>
+
-
<ul>
+
-
<li>buffer used: 4 ; Restriction-enzymes used: Enzyme 1: NotI; Enzyme 2: PstI</li>
+
-
<li>Plasmid
+
-
<ul>
+
-
<li>Plasmid-Number: from Sven; DNA concentration: 259 ng/µL ;</li>
+
-
</ul>
+
-
</ul>
+
-
<br />
+
<br />
<br />
{| border="1"
{| border="1"
-
| align="left" | '''Components''' ||align="left"| '''Volume/µL'''  
+
| ''' '''  || align="right" |'''vector /µl''' || align="right" |'''insert /µl'''
|-
|-
-
| align="left" | DNA ||align="left"| 3.8
+
| pSB1C3_BLA || align="right" |0,86 || align="right" |7,14
|-
|-
-
| align="left" | BSA (10x) ||align="left"| 1.5
 
-
|-
 
-
| align="left" | Buffer 4 (10x) ||align="left"| 1.5
 
-
|-
 
-
| align="left" | NotI-HF ||align="left"| 0.75
 
-
|-
 
-
| align="left" | PstI-HF ||align="left"| 0.75
 
-
|-
 
-
| align="left" | H<sub>2</sub>O ||align="left"| 3.2
 
-
|-
 
-
| align="left" | <b>Total volume</b> ||align="left"| <b>15</b>
 
|}
|}
<br />
<br />
-
Incubation: 1 h
 
-
<br />
 
-
<p style="font-size:15px; font-weight: bold; color:#ff00ff;">Agarose-Gel:</p>
 
-
<br />
 
-
0.5  g Agarose, 50 mL TBE (1 %),3 µL GELRED, at 115 Volt, running time: 45 minutes
 
-
<br />
 
-
<br />
 
-
{| border="1" cellspacing="0" cellpadding="2" bordercolor="black"
 
-
!Sample
 
-
!Sample/µl]
 
-
!Loading dye (5x)/µl
 
-
!Expected size <b>ITRs</b>
 
-
!Expected size 2
 
-
!Expected size 3
 
-
!Expected size 4
 
-
|--
 
-
|pAAV_MCS
 
-
|15 µl
 
-
|2 µl
 
-
|145 bp
 
-
|1216 bp
 
-
|555 bp
 
-
|2608 bp
 
-
|--
 
-
|}
 
-
{| align=right
 
-
|}
 
-
<br />
 
-
*Marker: GeneRuler ladder mix
 
-
{| border="1"
 
-
|
 
-
!Marker /µL
 
-
!Sample /µl
 
-
!Marker /µL
 
-
|-
 
-
!Lane
 
-
|6.5
 
-
|12
 
-
|6.5
 
-
|-
 
-
|}
 
-
<br />
 
-
[[File:Freiburg10 testDigestion pAAV MCS.jpg|700px]] <br/>
 
-
<p style="font-size:15px; color:#ff00ff;"><b>Comments:</b> The test digestion showed that there're two bands between the 100 and 200 bp marker nucleotides! By comparing the gel picture with the last mini-prep digestion of the fancy method we found out that the space between the two bands are similar. Because of that we assumed that the 15 pb are already missing in the original Stratagene plasmid!!!  </p> <br/>
+
*Transformation:
-
<p style="font-size:15px; color:#800080;"><b>Conclusion:</b> Because we already tested the Stratagene Kit via YFP expression, we can conclude that the usage of the "wrong" rigth ITR works. Therefore we decided to continue working with this "mutated" = <b> ENGINEERED </b> :) right ITR as BioBrick.</p> <br/>
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Miniprep of pSBC13_Rep78, pSB1C3_Rep68, pSB1C3_AAP, pSB1C3_rightITR and psB1C3_leftITR</b></p>====
+
The transformation was done following the standard protocol using XL1 blue cells.<br />
 +
<br />
-
<p style="font-size:13px; color: blue;">Plasmid Mini-Prep</p>
+
<p style="font-size:13px; color:#003399;"><b>Comment</b>: Two clones were picked, but because we already obtained our plasmid via SDM in the meantime, no prep was performed. Glycerol stocks of the two clones were created just in case, B264 and B265.</p>
-
Investigator: Kerstin<br>
+
-
<br>
+
 +
====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-Preps and test digestion of pSB1C3_Affibody_Middle-Linker, pSB1C3_Affibody_Short-Linker, pSB1C3_Affibody_SEG-Linker, pSB1C3_Affibody_Long-Linker and pSB1C3_betaglobin_mVenus_hGH_rITR</b></p>====
 +
<b>Investigator: Stefan</b><br>
 +
<ul>
 +
<b>Glycerol stocks were prepared:</b>
 +
<li>B240 = pSB1C3_Affibody_Middle-Linker clone1</li>
 +
<li>B241 = pSB1C3_Affibody_Middle-Linker clone2</li>
<br />
<br />
-
<u><b>Glycerol Stocks</b></u> <br>
 
-
<b>pSB1C3_Rep78</b>
 
-
{| border="1"
 
-
| align="left" |  ||align="left"| '''Clone 1''' ||align="left"| '''Clone 2'''
 
-
|-
 
-
| align="left" | '''Bacteria strain''' ||align="left"| BL-21 ||align="left"| BL-21
 
-
|-
 
-
| align="left" | '''given glycerol-stock no.''' ||align="left"| B128 ||align="left"| B129
 
-
|-
 
-
| align="left" | '''given plasmid no.''' ||align="left"| P160 ||align="left"| p161
 
-
|-
 
-
| align="left" | '''DNA-concentration ng/µl''' ||align="left"| 139,1 ||align="left"| 71,0
 
-
|}
 
-
<b>pSB1C3_2_Rep78</b>
 
-
{| border="1"
 
-
| align="left" |  ||align="left"| '''Clone 1''' ||align="left"| '''Clone 2'''
 
-
|-
 
-
| align="left" | '''Bacteria strain''' ||align="left"| BL-21 ||align="left"| BL-21
 
-
|-
 
-
| align="left" | '''given glycerol-stock no.''' ||align="left"| B130 ||align="left"| B131
 
-
|-
 
-
| align="left" | '''given plasmid no.''' ||align="left"| P162 ||align="left"| p163
 
-
|-
 
-
| align="left" | '''DNA-concentration ng/µl''' ||align="left"| 112,1 ||align="left"| 79,7
 
-
|}
 
-
<b>pSB1C3_Rep68</b>
 
-
{| border="1"
 
-
| align="left" |  ||align="left"| '''Clone 1''' ||align="left"| '''Clone 2'''
 
-
|-
 
-
| align="left" | '''Bacteria strain''' ||align="left"| BL-21 ||align="left"| BL-21
 
-
|-
 
-
| align="left" | '''given glycerol-stock no.''' ||align="left"| B132 ||align="left"| B133
 
-
|-
 
-
| align="left" | '''given plasmid no.''' ||align="left"| P164 ||align="left"| p165
 
-
|-
 
-
| align="left" | '''DNA-concentration ng/µl''' ||align="left"| 80,2 ||align="left"| 72,8
 
-
|}
 
-
<b>pSB1C3_2_Rep68</b>
 
-
{| border="1"
 
-
| align="left" |  ||align="left"| '''Clone 1''' ||align="left"| '''Clone 2'''
 
-
|-
 
-
| align="left" | '''Bacteria strain''' ||align="left"| BL-21 ||align="left"| BL-21
 
-
|-
 
-
| align="left" | '''given glycerol-stock no.''' ||align="left"| B134 ||align="left"| B135
 
-
|-
 
-
| align="left" | '''given plasmid no.''' ||align="left"| P166 ||align="left"| p167
 
-
|-
 
-
| align="left" | '''DNA-concentration ng/µl''' ||align="left"| 85,6 ||align="left"| 69,1
 
-
|}
 
-
<b>pSB1C3_AAP</b>
 
-
{| border="1"
 
-
| align="left" |  ||align="left"| '''Clone 1''' ||align="left"| '''Clone 2'''
 
-
|-
 
-
| align="left" | '''Bacteria strain''' ||align="left"| BL-21 ||align="left"| BL-21
 
-
|-
 
-
| align="left" | '''given glycerol-stock no.''' ||align="left"| B136 ||align="left"| B137
 
-
|-
 
-
| align="left" | '''given plasmid no.''' ||align="left"| P168 ||align="left"| p169
 
-
|-
 
-
| align="left" | '''DNA-concentration ng/µl''' ||align="left"| 63,6 ||align="left"| 56,3
 
-
|}
 
-
<b>pSB1C3_2_AAP</b>
 
-
{| border="1"
 
-
| align="left" |  ||align="left"| '''Clone 1''' ||align="left"| '''Clone 2'''
 
-
|-
 
-
| align="left" | '''Bacteria strain''' ||align="left"| BL-21 ||align="left"| BL-21
 
-
|-
 
-
| align="left" | '''given glycerol-stock no.''' ||align="left"| B138 ||align="left"| B139
 
-
|-
 
-
| align="left" | '''given plasmid no.''' ||align="left"| P170 ||align="left"| p171
 
-
|-
 
-
| align="left" | '''DNA-concentration ng/µl''' ||align="left"| 135,1 ||align="left"| 114,2
 
-
|}
 
-
<b>pSB1C3_rightITR</b>
 
-
{| border="1"
 
-
| align="left" |  ||align="left"| '''Clone 1''' ||align="left"| '''Clone 2'''
 
-
|-
 
-
| align="left" | '''Bacteria strain''' ||align="left"| BL-21 ||align="left"| BL-21
 
-
|-
 
-
| align="left" | '''given glycerol-stock no.''' ||align="left"| B140 ||align="left"| B141
 
-
|-
 
-
| align="left" | '''given plasmid no.''' ||align="left"| P172 ||align="left"| p173
 
-
|-
 
-
| align="left" | '''DNA-concentration ng/µl''' ||align="left"| 73,7 ||align="left"| 66,4
 
-
|}
 
-
<b>pSB1C3_leftITR</b>
 
-
{| border="1"
 
-
| align="left" |  ||align="left"| '''Clone 1''' ||align="left"| '''Clone 2'''
 
-
|-
 
-
| align="left" | '''Bacteria strain''' ||align="left"| BL-21 ||align="left"| BL-21
 
-
|-
 
-
| align="left" | '''given glycerol-stock no.''' ||align="left"| B142 ||align="left"| B143
 
-
|-
 
-
| align="left" | '''given plasmid no.''' ||align="left"| P174 ||align="left"| p175
 
-
|-
 
-
| align="left" | '''DNA-concentration ng/µl''' ||align="left"| 74,9 ||align="left"| 80,3
 
-
|}
 
-
<br>
 
-
'''Test digestion:'''<br>
 
-
pSB1C3_Rep78, pSB1C3_Rep68 and pSB1C3_AAP were digested with XbaI and SpeI<br>
 
-
pSB1C3_rightITR and pSB1C3_leftITR were digested with EcoRI and PstI.<br>
 
-
<br>
+
<li>B242 = pSB1C3_Affibody_Short-Linker clone 1</li>
-
{| border="1"
+
<li>B243 = pSB1C3_Affibody_Short-Linker clone 2</li>
-
| components  || align="right" |volume of '''P160'''/µl ||volume of '''P161'''/µl||volume of '''P162/'''µl ||volume of '''P163'''/µl ||volume of '''P164'''/µl||volume of '''P165'''/µl||volume of '''P166'''µl ||volume of '''P167'''/µl||volume of '''P168'''µl ||volume of '''P169'''/µl||volume of '''P170'''/µl||volume of '''P171'''/µl||volume of '''P172'''/µl||volume of '''P173'''µl ||volume of '''P174'''/µl ||volume of '''P175'''/µl
+
-
|-
+
-
| DNA  ||5,8 ||11,3 ||7,1 ||10 ||9,9 ||10,9 ||9,3 ||11,6 ||12,5 ||14,2 ||5,9 ||7,0 ||10,9 ||12,0 ||10,7 ||9,7
+
-
|-
+
-
| BSA (10x) ||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2
+
-
|-
+
-
| Buffer 4 (10x)||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2 ||2
+
-
|-
+
-
|Enzyme 1 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 
+
-
|-
+
-
|Enzyme 2 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5 ||0,5
+
-
|-
+
-
|H2O ||9,2 ||3,7 ||7,9 ||5,0 ||5,1 ||4,1 ||5,7 ||3,4 ||2,5 ||0,8 ||9,1 ||8 ||4,1 ||3 ||4,3 ||5,3
+
-
|-
+
-
|'''Total volume /µl'''||20 ||20 ||20 ||20 ||20 ||20 ||20 ||20 ||20 ||20 ||20 ||20 ||20 ||20 ||20 ||20
+
-
|}
+
<br />
<br />
-
'''Expected sizes:'''<br>
 
-
*Rep78: 1,8 kb
 
-
*Rep68: 1,6kb<br>
 
-
*AAP: 650bp <br>
 
 +
<li>B246 = pSB1C3_Affibody_SEG-Linker clone1</li>
 +
<li>B247 = pSB1C3_Affibody_SEG-Linker clone2</li>
 +
<br />
-
*left ITR: 150bp
+
<li>B248 = pSB1C3_Affibody_Long-Linker clone 1</li>
-
*right ITR: 150bp
+
<li>B249 = pSB1C3_Affibody_Long-Linker clone 2</li>
 +
<br />
-
<br>
+
<li>B244 = pSB1C3_betaglobin_mVenus_hGH_rITR clone 1</li>
-
<p style="font-size:13px; color:#11bb88;">'''Results of samples loaded 1,5% agarose gel and ran ~50 minutes at 115 V:''' </p>
+
<li>B245 = pSB1C3_betaglobin_mVenus_hGH_rITR clone 2</li>
-
[[File:Freiburg10 test digestion itr und aap.jpg|400px]]
+
<br />
-
<br>
+
-
<p style="font-size:13px; color:#11bb88;">'''Results of samples loaded 0,8% agarose gel and ran ~50 minutes at 115 V:''' </p>
+
-
[[File:Freiburg10 test digestion rep 78 u. 68.jpg|400px]]
+
 +
<b>Mini-Prep following the standard protocol</b>
 +
<li>P290 = pSB1C3_Affibody_Middle-Linker clone1 c= 227,36 ng/µl</li>
 +
<li>P291 = pSB1C3_Affibody_Middle-Linker clone2 c= 219,05 ng/µl</li>
 +
<br />
-
<p style="font-size:13px; color:#11bb56;">'''Sequencing''' </p>
+
<li>P292 = pSB1C3_Affibody_Short-Linker clone 1 c= 196,63 ng/µl</li>
-
P160, P165 and P170 were send for sequencing.
+
<li>P293 = pSB1C3_Affibody_Short-Linker clone 2 c= 224,76 ng/µl</li>
-
===82.Labortag 07.08.2010===
+
<br />
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Cellculture</b></p>====
+
<li>P296 = pSB1C3_Affibody_SEG-Linker clone1 c= 218,68 ng/µl</li>
 +
<li>P297 = pSB1C3_Affibody_SEG-Linker clone2 c= 229,66 ng/µl</li>
 +
<br />
-
HEK 293 cells were split and seeded according to the standard protocol: 2x T75 flask and 10x10cm cellculture dishes. Investigator: Patrick
+
<li>P298 = pSB1C3_Affibody_Long-Linker clone 1 c= 229,73 ng/µl</li>
 +
<li>P299 = pSB1C3_Affibody_Long-Linker clone 2 c= 202,05 ng/µl</li>
 +
<br />
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Continuation: Cloning Rep40 & Rep52 into pSB1C3</b></p>====
+
<li>P294 = pSB1C3_betaglobin_mVenus_hGH_rITR clone 1 c= 425,14 ng/µl</li>
-
Investigator: Patrick <br>
+
<li>P295 = pSB1C3_betaglobin_mVenus_hGH_rITR clone 2 c= 328,28 ng/µl</li>
-
Results: no clones could be picked because there were no clones. Maybe the PCR purification or one of the following steps did not work (the PCR purifications were quite bad) so i purified the PCR products again yielding very low DNA concentrations (0-13,28 ng/µl) and still very obvious pollution of the sample. Nonetheless a ligation with the insert Rep40+DMSO, Rep52, Rep52+DMSO and the vector pSB1C3 was carried out following a transformation and according to the standard protocols. Hopefully there will be some clones tomorrow.
+
-
 
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-Prep and test digestion of pSB1C3_SDM_SspI/PvuII</b></p>====
+
-
'''Investigator: Jessica'''<br>
+
-
<br>
+
-
<p style="font-size:18px; font-weight: bold; color:#66bbff;"><u>Plasmid Mini-Prep</u></p>
+
-
*new vector name: pSB1C3_SDM_SspI/PvuII
+
<br />
<br />
-
<u><b>Glycerol Stocks</b></u> <br>
+
</ul>
-
<b>pSB1C3_SDM_SspI/PvuII</b>
+
<b>Test digestion</b><br>
 +
<ul>
 +
<li>Restriction-enzymes used: for P290-P293 and P296-P299: NotI ; for P294-P295: NgoMIV and PstI </li>
 +
</ul><br />
 +
<b>'''comment:'''</b> The same amount of ingredients were used for P290-P293 and P296-P299, therefore these approaches will be merged into the chart. The same goes for P294-P295.
{| border="1"
{| border="1"
-
| align="left" |  ||align="left"| '''Clone 1''' ||align="left"| '''Clone 2''' ||align="left"| '''Clone 3'''  
+
| align="left" | '''Components''' ||align="left"| '''P290-P293 and P296-P299''' ||align="left"| '''P294-P295'''  
|-
|-
-
| align="left" | '''Bacteria strain''' ||align="left"| BL21 ||align="left"| BL21  ||align="left"| BL21   
+
| align="left" | DNA ||align="left"| 1 ||align="left"| 1
|-
|-
-
| align="left" | '''Plasmidname''' ||align="left"| pSb1C3_SDM_SspI/PvuII ||align="left"| pSb1C3_SDM_SspI/PvuII ||align="left"| pSb1C3_SDM_SspI/PvuII
+
| align="left" | BSA (10x) ||align="left"| 1 ||align="left"| 1
|-
|-
-
| align="left" | '''Date''' ||align="left"| 7.8.10 ||align="left"| 7.8.10 ||align="left"| 7.8.10 
+
| align="left" | Buffer 4 (10x) ||align="left"| 1 ||align="left"| 1
|-
|-
-
| align="left" | '''given glycerol-stock no.''' ||align="left"| B144 ||align="left"| B145 ||align="left"| B146
+
| align="left" | NotI ||align="left"| 0,5 ||align="left"| -
|-
|-
-
| align="left" | '''given plasmid no.''' ||align="left"| P157.1 ||align="left"| P157.2 ||align="left"| P157.3 
+
| align="left" | NgoMIV ||align="left"| - ||align="left"| 0,5
-
|}
+
-
<br>
+
-
 
+
-
 
+
-
<p style="font-size:15px; font-weight: bold; color:#66bbff;">Test digestion</p>
+
-
*buffer used: 2 ; Restriction-enzymes used: Enzyme 1 SspI ; Enzyme 2 PvuII
+
-
*Plasmid: pSB1C3_SDM_SspI/PvuII:
+
-
**Given Plasmid-Number: P157.1; DNA concentration: 246,3 ng/µL ;
+
-
**Given Plasmid-Number: P157.2; DNA concentration: 231,7 ng/µL ;
+
-
**Given Plasmid-Number: P157.3; DNA concentration: 219,6 ng/µL ;
+
-
<br />
+
-
 
+
-
{| border="1"
+
-
| align="left" | '''Components''' ||align="left"| <b>P157.1</b> Volume/µL ||align="left"| <b>P157.2</b> Volume/µL ||align="left"| <b>P157.3</b> Volume/µL
+
|-
|-
-
| align="left" | DNA ||align="left"| 2,8 ||align="left"| 3,0 ||align="left"| 3,19
+
| align="left" | PstI ||align="left"| - ||align="left"| 0,5
|-
|-
-
| align="left" | BSA (10x) ||align="left"| - ||align="left"| - ||align="left"| -
+
| align="left" | H<sub>2</sub>O ||align="left"| 6,5 ||align="left"| 6
|-
|-
-
| align="left" | Buffer no. 2 (10x) ||align="left"| 1 ||align="left"| 1 ||align="left"| 1
+
| align="left" | <b>Total volume</b> ||align="left"| <b>10</b> ||align="left"| <b>10</b>  
-
|-
+
-
| align="left" | Enzyme 1 SspI ||align="left"| 0.5 ||align="left"| 0.5 ||align="left"| 0.5
+
-
|-
+
-
| align="left" | Enzyme 2 PvuII ||align="left"| 0.5 ||align="left"| 0.5 ||align="left"| 0.5
+
-
|-
+
-
| align="left" | H<sub>2</sub>O ||align="left"| 5,2 ||align="left"| 5 ||align="left"| 4,81
+
-
|-
+
-
| align="left" | '''Total volume''' ||align="left"| <b>10</b> ||align="left"| <b>10</b> ||align="left"| <b>10</b>
+
|}
|}
<br />
<br />
-
 
+
Incubation time: 70 minutes; Incubation temperature: 37°
-
*Incubation: 45 min
+
<br />
<br />
-
<p style="font-size:15px; font-weight: bold; color:#66bbff;">Agarose-Gel:</p>
+
0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , running time:5 minutes at 90 Volt and 50 minutes at 115 Volt <br />
 +
2µl loading dye (6x) for the sample, Marker: GeneRuler ladder mix (Fermentas)
<br />
<br />
-
0.45 g Agarose, 50 mL <b>TAE</b>, 3 µL GELRED, at 115 Volt, running time: 45 minutes
 
<br />
<br />
 +
[[File:Freiburg10 test digestion affi+linker right assembly.jpg|500px]]
<br />
<br />
-
{| border="1" cellspacing="0" cellpadding="2" bordercolor="black"
 
-
!Sample
 
-
!Sample/µl]
 
-
!Loading dye (6x)/µl
 
-
 
-
|--
 
-
|P157.1
 
-
|10 µl
 
-
|1,6 µl
 
-
 
-
|--
 
-
|P157.2
 
-
|10 µl
 
-
|1,6 µl
 
-
 
-
|--
 
-
|P157.3
 
-
|10 µl
 
-
|1,6 µl
 
-
 
-
|--
 
-
 
-
|}
 
-
{| align=right
 
-
|}
 
<br />
<br />
-
*Marker: GeneRuler ladder mix
+
<p style="font-size:13px; color:#003399;"><b>Comment</b>: Sizes of fragments look like expected. Clone 1 of each plasmid will be sent for sequencing tomorrow.</p>
-
{| border="1"
+
-
|
+
-
!Marker /µL
+
-
!Sample P157.1 /µL
+
-
!Sample P157.2 /µL
+
-
!Sample P157.3 /µL
+
-
|-
+
-
!Lane
+
-
|7
+
-
|10
+
-
|10
+
-
|10
+
-
|-
+
-
|}
+
-
<br />
+
-
<br>
+
-
I expect one cut from the PvuII RS in the CFP --> linearized <br>
+
-
[[File:Prep pSB1C3_SDM_SspIPvuII.jpg|400px]] <br>
+
-
<br>
+
-
<b>Comment:</b><br> 
+
-
<br>
+
-
<p style="font-size:13px; color:#ff0000;">'''''Comments:'''''Oh f... it makes no sense... if the cloning didn't work, there can just be 3 bands (one time SspI and twice PvuII). The results of the test digestion from aug 1th showed that there can be one RS PvuII more. but then you have a maximum of 4 bands. '''P157.2''' show '''5''' bands. i can't interpret this result at the moment. interpretation will follow</p> <br>
+
-
<br/>
+
-
<br/>
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Evaluation of test transformation with XL1 blue cells and pUCI</b></p>====
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Sequenzing evaluation of SDM NgoMIV</b></p>====
-
'''Investigator: Kira'''<br>
+
<b>Investigator: Kira</b><br>
 +
 +
2 samples have been sent for sequencing yesterday. According to the data, both samples do not contain any NgoMIV restriction site anymore. <br />
-
Transformation was performed with lab XL1B and iGEM XL1B cells. iGEM plate contained just 25 colonies, while lab plate contained around 50 colonies.
 
-
Conclusion: the production of competent cells has to be repeated.
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Biobrick production of p5 promoter from pTAV2 and pAAV_RC</b></p>====
 
-
'''Investigator: Kira'''<br>
 
-
*'''PCR:
 
-
DNA samples were diluted 1:100
+
[[File:Freiburg10 sample 1, seq 1.jpg|500px]]
-
<br>
 
-
{| border="1"
 
-
| '''Ingredients''' || align="right" |'''p 32''' ||align="right"|p 50
 
-
|-
 
-
| 5X Phusion HF buffer ||  align="right" |10 µl||align="right"|10 µl
 
-
|-
 
-
| 10 mM dNTP mix||  align="right" |1µl ||align="right"|1 µl
 
-
|-
 
-
| forward primer: O114 ||  align="right" |2,5µl||align="right"|2,5 µl
 
-
|-
 
-
| reverse primer: O115 ||  align="right" |2,5 µl||align="right"|2,5 µl
 
-
|-
 
-
| DNA Template||  align="right" |0,3 µl||align="right"| 0,4 µl
 
-
|-
 
-
| DMSO ||  align="right" |0 µl||align="right"|0 µl
 
-
|-
 
-
| Phusion Polymerase||  align="right" |0,5 µl||align="right"| 0,5 µl
 
-
|-
 
-
|H<sub>2</sub>O||  align="right" |33,2 µl||align="right"|28,6 µl
 
-
|-
 
-
|Total volume||  align="right" |50 µl||align="right"| 50 µl
 
-
|}
 
-
<br>
 
-
PCR program:
+
[[File:Freiburg10 sample 3, seq2.jpg|500px]]
-
{| border="1"
+
===101. labday 26.08.2010===
-
|Cycles||Temperature||Time
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-Preps of pGA14_middle linker and pSB1C3_6xHis clone 1</b></p>====
-
|-
+
<b>Investigator: Jessica</b><br>
-
|||98°C||1
+
<ul>
-
|-
+
Glycerol stocks were not prepared because I have inoculated from glycerol stocks
-
|8x||98°C||30"
+
-
|-
+
-
|||60°C||25"
+
-
|-
+
-
|||72°C||6"
+
-
|-
+
-
|17x||98°C||30"
+
-
|-
+
-
|||70°C||25"
+
-
|-
+
-
|||72°C||6"
+
-
|-
+
-
|1x||72°C||5'
+
-
|-
+
-
|Hold 4°C
+
-
|}
+
<br>
<br>
-
Digestion of plasmid backbone:
+
<b>Mini-Prep following the standard protocol</b>
-
 
+
<li>P301 = pGA14_middle linker (= '''P65''')<br>
-
c (pSB1C3) = 151, 1 ng/ µl
+
c=305,7</li>
-
 
+
-
{| border="1"
+
-
| align="left" | '''Components''' ||align="left"| <b>vector</b> Volume/µL
+
-
|-
+
-
| align="left" | DNA 1 µg  ||align="left"| 6,0 µl
+
-
|-
+
-
| align="left" | BSA (100x) ||align="left"| 0,2 µl
+
-
|-
+
-
| align="left" | Buffer no. 4 (10x) ||align="left"| 2,0 µl
+
-
|-
+
-
| align="left" | Enzyme 1 XbaI ||align="left"| 1 µl
+
-
|-
+
-
| align="left" | Enzyme 2 PstI HF ||align="left"| 1 µl
+
-
|-
+
-
| align="left" | H<sub>2</sub>O ||align="left"| 9,8 µl
+
-
|-
+
-
| align="left" | '''Total volume''' ||align="left"| <b>20</b>
+
-
|}
+
<br />
<br />
 +
<li>P300 = pSB1C3_6xHis clone 1 (= '''P84''')<br>
 +
c=177,5</li>
-
incubation @ 37 C for approx. 2 h
 
-
 
-
1% agarose gel with PCR products:
 
-
 
-
[[File:Freiburg10 PCR.jpg|400px]]
 
<br />
<br />
 +
</ul>
-
Concentrations after gel extraction:
 
-
c(p32) = 67,95 ng/µl <br />
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-Preps and test digestion of pAAV_RC-ins_rep_cap, pCeruleanVP1up and pAAV_RC_ins-rep-cap.</b></p>====
-
c(p50) = 32,45 ng/µl <br />
+
-
c(vector) = 7,54 ng/µl <br />
+
-
<br />
+
-
Digestion of PCR products:
 
-
{| border="1"
 
-
| align="left" | '''Components''' ||align="left"| <b>PCR product</b> Volume/µL
 
-
|-
 
-
| align="left" | DNA 1 µg  ||align="left"| 23,0 µl
 
-
|-
 
-
| align="left" | BSA (100x) ||align="left"| 0,3 µl
 
-
|-
 
-
| align="left" | Buffer no. 4 (10x) ||align="left"| 3,0 µl
 
-
|-
 
-
| align="left" | Enzyme 1 XbaI ||align="left"| 1,5 µl
 
-
|-
 
-
| align="left" | Enzyme 2 PstI HF ||align="left"| 1,5 µl
 
-
|-
 
-
| align="left" | H<sub>2</sub>O ||align="left"| 0,7 µl
 
-
|-
 
-
| align="left" | '''Total volume''' ||align="left"| <b>30</b> 
 
-
|}
 
-
<br />
 
-
Concentrations after PCR purification:
+
<b>Investigator: Chris L.</b><br>
-
 
+
<ul>
-
c(p32) = 21, 63 ng/µl <br />
+
<b>Glycerol stocks were prepared:</b>
-
c(p50) = 19,48 ng/µl <br />
+
<li>B250 = pCerulean_VP1up clone1</li>
 +
<li>B251 = pCerulean_VP1up clone2</li>
 +
<li>B252 = pCerulean_VP1up clone3</li>
 +
<li>B253 = pCerulean_VP1up clone4</li>
 +
<li>B254 = pSB1C3_VCK_Bla clone1</li>
 +
<li>B255 = pSB1C3_VCK_Bla clone2</li>
 +
<li>B256 = pSB1C3_VCK_Bla clone3</li>
 +
<li>B257 = pSB1C3_VCK_Bla clone4</li>
 +
<li>B262 = pAAV_RC_ins-rep-cap clone1</li>
 +
<li>B263 = pAAV_RC_ins-rep-cap clone2</li>
<br />
<br />
-
 
+
<b>Mini-Prep following the standard protocol</b>
-
Ligation:
+
<li>P302 = pCerulean_VP1up clone1<br>c=410,70 ng/µl</li>
-
 
+
<li>P303 = pCerulean_VP1up clone2<br>c=387,78 ng/µl</li>
-
Quickligase was used for ligation.
+
<li>P304 = pCerulean_VP1up clone3<br>c=321,04 ng/µl</li>
-
 
+
<li>P305 = pCerulean_VP1up clone4<br>c=350,34 ng/µl</li>
-
p32 DNA-Mix: 8 µl vector + 1 µl insert <br />
+
<li>P306 = pSB1C3_VCK_Bla clone1<br>c=82,53 ng/µl</li>
-
p50 DNA-Mix: 7,9 µl vector + 1,1 µl insert <br />
+
<li>P307 = pSB1C3_VCK_Bla clone2<br>c=92,70 ng/µl</li>
 +
<li>P308 = pSB1C3_VCK_Bla clone3<br>c=82,22 ng/µl</li>
 +
<li>P309 = pSB1C3_VCK_Bla clone4<br>c=105,34 ng/µl</li>
 +
<li>P314 = pAAV_RC_ins-rep-cap clone1<br>c=517,04 ng/µl</li>
 +
<li>P315 = pAAV_RC_ins-rep-cap clone2<br>c=444,33 ng/µl</li>
<br />
<br />
-
Transformation:
+
</ul>
-
 
+
<b>Test digestion:</b>
-
Transformation was performed according to the standard protocol with DYT and BL21 cells. The cells were spread on the agar plates containing chloramphenicol.
+
-
<br />
+
-
 
+
-
 
+
-
====<p style="font-size:15px; background-color:#ff00ff;">'''Sequencing results of SalI SDM'''</p>====
+
-
Investigator: <b>Hanna</b>
+
-
<br/>
+
-
 
+
-
<p style="color:#ff00ff;"><b>Comment:</b> The test digestion already showed that the SDM of the SalI restriction site in pAAV_RC worked. This could be validated by sequencing the plasmid with the Rep-1250_for primer: <br/> </p>
+
-
<br/>
+
-
[[File:Freiburg10 SalI.jpg|800px]]
+
-
<br/>
+
-
<br/>
+
-
 
+
-
P58 can be used for further RepCap-experiments.
+
-
 
+
-
===83. Labortag 08.08.2010===
+
-
 
+
-
HT 1080 cells were split and seeded: 3x10^6 cells per into each well of a 6-well plate.
+
-
 
+
-
A431 cells were washed and received new medium.
+
-
 
+
-
There were no clones on the agar plates so they were put back into the 37°C room. Maybe there will be some clones tomorrow.
+
-
 
+
-
===84. Labortag 09.08.2010===
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Midi-Prep Inoculation</b></p>====
+
-
*pAAV_RC
+
-
*pAAV_RC_1.2 SDM SalI (P158)
+
-
*pAAV_RC_mGMK_TK30 clone 1 (P81)
+
-
*pAAV_RC_mGMK_TK30 clone 2 (P82)
+
-
 
+
-
Investigators: Chris W., Patrick
+
-
 
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Continuation: Cloning Rep40 & Rep52 into pSB1C3</b></p>====
+
-
<b>Investigator: Patrick </b> <br />
+
-
Two Rep52+DMSO clones grew. They were picked and tomorrow there will be a midi prep and a test digestion.
+
-
Simultaneously a new approach for cloning Rep40 and Rep52 into pSB1C3 was performed. (see next header)
+
-
 
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Repetition: BioBrick production of Rep40 & Rep52</b></p>====
+
-
 
+
-
'''Investigator: Anna
+
-
 
+
-
<p style="font-size:13px; color:#68bbff;">'''''Comments''''': Another approach was done because there were no clones for Rep 40 and only two for Rep 52 (Cloning see 06.08.2010). PCR was performed again with DMSO. Samples from gel extraction are stored in 4°C freezer. </p> <br>
+
<br>
<br>
-
'''PCR
 
-
 
{| border="1"
{| border="1"
-
| '''Ingredients''' || align="right" |'''Rep 40''' ||align="right"|'''Rep52'''
+
| components  || align="right" |volume of '''P302'''/µl ||volume of '''P303'''/µl||volume of '''P304/'''µl ||volume of '''P305'''/µl ||volume of '''P306'''/µl||volume of '''P306'''/µl||volume of '''P306'''µl ||volume of '''P307'''/µl||volume of '''P307'''µl ||volume of '''P307'''/µl||volume of '''P308'''/µl||volume of '''P308'''/µl ||volume of '''P308'''/µl ||volume of '''P309'''/µl ||volume of '''P309'''/µl ||volume of '''P309'''/µl||volume of '''P314'''/µl ||volume of '''P315'''/µl
|-
|-
-
| 5X Phusion HF buffer || align="right" |10 µl||align="right"|10 µl
+
| DNA  || 1 || 1 || 1 || 1 || 2 || 2 || 2 || 2 || 2 || 2 || 2 || 2 || 2 || 2 || 2 || 2 || 1 || 1
|-
|-
-
| 10 mM dNTP mix|| align="right" |1µl ||align="right"|1 µl
+
| BSA (10x) ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1  
|-
|-
-
| forward primer: O094 || align="right" |2,5µl||align="right"|2,5 µl
+
| Buffer 4 (10x) ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||1 ||- ||-
|-
|-
-
| reverse primer: O0096/ O097 || align="right" |2,5 µl||align="right"|2,5 µl
+
| Buffer 2 (10x) ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||-||- ||1 ||1
|-
|-
-
| DNA Template (1ng)|| align="right" |1 µl||align="right"| 1 µl
+
|Enzyme NotI ||- ||- ||- ||- ||0,5 ||- ||- ||0,5 ||- ||- ||0,5 ||- ||- ||0,5 ||- ||- ||- ||-
|-
|-
-
| DMSO || align="right" |1 µl||align="right"|1 µl
+
|Enzyme SspI ||- ||- ||- ||- ||- ||0,5 ||- ||- ||0,5 ||- ||- ||0,5 ||- ||- ||0,5 ||- ||- ||-
|-
|-
-
| Phusion Polymerase|| align="right" |0,5 µl||align="right"| 0,5 µl
+
|Enzyme SalI ||- ||- ||- ||- ||- ||- ||0,5 ||- ||- ||0,5 ||-||- ||0,5 ||- ||- ||0,5 ||- ||-
|-
|-
-
|H<sub>2</sub>O|| align="right" |31,5 µl||align="right"|31,5 µl
+
|Enzyme BamHI ||- ||- ||- ||- ||- ||- ||0,5 ||- ||- ||- ||- ||- ||- ||- ||- ||- ||-||-
|-
|-
-
|Total volume|| align="right" |50 µl||align="right"| 50 µl
+
|Enzyme PvuII ||- ||- ||- ||- ||- ||- ||0,5 ||- ||- ||- ||- ||- ||- ||- ||- ||- ||-||-
-
|}
+
-
<br>
+
-
 
+
-
'''PCR programm
+
-
 
+
-
{| border="1"
+
-
|'''PCR Program'''|| align="right" |'''temperature/ °C'''|| align="right" |'''Time''' || align="right" |'''PCR products
+
|-
|-
-
|1|| align="right" |98 || align="right" |1min|| align="right" |
+
|Enzyme Acc65I ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||-||- ||0,75 ||0,75
|-
|-
-
|2|| align="right" |98 || align="right" |15s|| align="right" |  
+
|Enzyme XcmI ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||-||- ||0,5 ||0,5
|-
|-
-
|8x|| align="right" |*** || align="right" |15s|| align="right" |63°C
+
|Enzyme PstI ||0,5 ||0,5 ||0,5 ||0,5 ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||-
|-
|-
-
|3 || align="right" |72|| align="right" |***|| align="right" |15s 
+
|Enzyme EcoRI ||0,5 ||0,5 ||0,5 ||0,5 ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||- ||-
|-
|-
-
|4 || align="right" |98|| align="right" | 15s|| align="right" |  
+
|H2O ||6 ||6 ||6 ||6 ||5,5 ||5 ||5 ||5,5 ||5 ||5 ||5,5 ||5||5 ||5,5 ||5 ||5 ||5,75 ||5,75
|-
|-
-
|19x|| align="right" |***|| align="right" |15s|| align="right" |68°C
+
|'''Total volume /µl'''||10 ||10 ||10 ||10 ||10 ||10 ||10 ||10 ||10 ||10 ||10 ||10 ||10 ||10 ||10 ||10 ||10 ||10
-
|-
+
-
|5|| align="right" |72|| align="right" |***|| align="right" |15s
+
-
|-
+
-
|6x|| align="right" |72|| align="right" |5min|| align="right" |
+
-
|-
+
-
|Hold|| align="right" |4||  align="right" |  ||  align="right" |  
+
|}
|}
-
<br>
 
-
 
-
'''Agarose gel
 
-
 
-
0.5  g Agarose, 50 mL TAE (1 %),3 µL GELRED, at 120 Volt, running time: 45 minutes
 
<br />
<br />
 +
Incubation time: 1 h, Incubation temperature: 37°
<br />
<br />
-
{| border="1" cellspacing="0" cellpadding="2" bordercolor="black"
+
Preparation of gel:<br />
-
!Sample
+
1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 50 minutes
-
!Sample / µl]
+
-
!Loading dye (6x) / µl
+
-
!Expected size
+
-
 
+
-
|--
+
-
|p22 (Rep40)
+
-
|44 µl
+
-
|7 µl
+
-
|940 bp
+
-
|--
+
-
|p23 (Rep52)
+
-
|44 µl
+
-
|7 µl
+
-
|~1200 bp
+
-
|--
+
-
|}
+
-
{| align=right
+
-
|}
+
-
<br />
+
-
*Marker: GeneRuler ladder mix
+
-
{| border="1"
+
-
|
+
-
!Marker /µL
+
-
!Sample Rep40 /µl
+
-
!Sample Rep52 /µl
+
-
|-
+
-
!Lane
+
-
|7
+
-
|51
+
-
|51
+
-
|-
+
-
|}
+
<br />
<br />
-
[[File:Freiburg10 Rep40 Rep52.png|800px]]<br />
+
[[File:Freiburg10 test digestion pSB1C3 VCK Bla+pCerulean Vp1up.jpg]]
-
'''Gel extraction<br/>
+
<b>Results:</b> <p style="color:#00bbff;">pSB1C3_VCK_Bla: Test digestion looks like expected. Clone 4 was sent for sequencing. <br />
-
PCR products were eluted with 20 µl BE puffer following the standard protocol.<br/>
+
pCerulean_VP1up: Gel looks well as well. clone 2 was sent for sequencing. <br/>
-
c(Rep40)= 28,03 ng/µl<br/>
+
pAAV_RC_ins-rep-cap: Test  digestion looks strange. Just one big band with more than 10000 bp and one very small with 150 bp. Maybe the restriction enzymes didn´t cut. (@Christian: please insert picture)</p>
-
c(Rep52)= 121,16 ng/µl<br/>
+
-
<p style="font-size:13px; color:#68bbff;">'''''Comments''''': Digestion was done with the wrong enzymes (EcoRI HF and SpeI), it will be repeated on 11.08. with XbaI and SpeI</p> <br>
+
===102. labday 27.08.2010===
-
 
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Sequence analysis of pSB1C3_Bla_final (P309)and pCerulean_VP1up (P303)</b></p>====
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Sequence analysis of pSB1C3_mGMK</b></p>====
+
<b>Investigator: Bea </b>
-
Investigator: <b>Bea </B>
+
<br />
 +
<p style="font-size:13px; color:#cc0033;"><I><b>GENERAL COMMENT:</b> pSB1C3_Bba_final (P309, SB_9) and pCerulean_Vp1up (P303; SB_10) were sent for sequencing. In the case of SB1C3_Bba_final, which means that the whole vector was assembled from the buttom,the purpose was to test the designed primers. In the other case we wanted to verify the insertion of Vp1up inot pCerulean. </i> </p>
<br />
<br />
-
<p style="font-size:13px; color:#003399;"><b>Comments</b>: mGMK was cloned into the pSB1C3 backbone in order to send it to the parts registry!. The sequence analysis revealed that the mGMK was <b>successfully </b> cloned into pSB1C3 plasmid. This polasmid is in the RFC25 standard.</p>  
+
<p style="font-size:13px; color:#003399;"><b>Comments</b>: Sequence analysis of pSB1C3_leftITR_CMV_betaglobin_mVenus_hGH_rightITR.</p>  
<ul>
<ul>
-
<b>Results sequencing: Sequence read ok! </b>
+
<li>Primer used: </li>
 +
<ul>
 +
<li>pSB1C3_Bba_seq_for</li>
 +
<li>pSB1C3_Bba_seq_rev</li>
 +
</ul>
 +
<li>Plasmid sequenced: P?? </li>
 +
<li>Sequence sample: ??</li>
 +
<li>Stored in Geneious-folder: BioBricks --> final parts</li>
 +
</ul>
 +
 
 +
<gallery widths=600px heights=400px perrow=2 caption="pSB1C3_leftITR_CMV_betaglobin_mVenus_hGH_rightITR">
 +
Image:Freiburg10 Seqanalysis pSB1C3 Bba final 2010 27 08.jpg
 +
</gallery>
<br />
<br />
-
Primer used: VR-2
+
<b>Results:</b> <p style="color:#00bbff;">Results look good. The forward primer worked, still waiting for the results of the reverse primer. The scar inbetween the CMV promoter and the beat globin intron corresponds to the expected result.</p>
 +
 
<br />
<br />
-
Comment: A mutation has been found in the pSB1C3 backone which was found generally in the backbone which we received from the iGEM HQ.
+
<b>Next steps: Wwait until sequencing results of the reverse primer can be confirmed aswell. If that will not be the case, order new sequensing primer.</b>
<br />
<br />
-
</ul>
 
-
[[File:Freiburg10 Sequence analysis of pSB1C3 mGMK 09 08 2010.jpg|1000px]]
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-Prep of pSB1C3_hTERT, pSB1C3_beta-globin_mVenus, pSB1C3_pTAV2(P5), pSB1C3_pAAV_RC(P5TATAles)</b></p>====
 
-
<b>Investigator: Jessica</b><br>
 
-
 
-
'''Bold text'''
 
-
Mini-Prep following the standart Protokoll
 
<br />
<br />
-
<li>P177 = pSB1C3_hTERT clone1 = 163,6 ng/µl
 
-
<li>P178 = pSB1C3_hTERT clone2 = 312,0 ng/µl
 
-
<li>P179 = pSB1C3_betaglobin_mVenus clone1 = 237,2 ng/µl
 
-
<li>P180 = pSB1C3_betaglobin_mVenus clone2 = 228,2 ng/µl
 
-
<li>P181 = pSB1C3_pTAV2(P5) clone1 = 108,8ng/µl
 
-
<li>P181 = pSB1C3_pTAV2(P5) clone2 = 140,9 ng/µl
 
-
<li>P181 = pSB1C3_pAAV_RC(P5TATAless) clone1 = 165,0 ng/µl
 
-
<li>P181 = pSB1C3_pAAV_RC(P5TATAless) clone2 = 176,6 ng/µl
 
-
<li>P181 = pSB1C3_pAAV_RC(P5TATAless) clone3 = 176,9 ng/µl
 
-
<br />
 
-
<br>
 
-
<br>
 
-
<b>Test digestion:</b>
 
-
<ul>
 
-
<li>Perfomed with 10 µL of total volume with all 9 clones</li>
 
-
<table border=1 cellpadding=0 cellspacing=0 width=621 style='border-collapse:
 
-
collapse;table-layout:fixed;width:466pt'>
 
-
<tr height=29 style='mso-height-source:userset;height:21.75pt'>
 
-
  <td height=29 class=xl6724191 width=143 style='height:21.75pt;width:107pt'>&nbsp;</td>
 
-
  <td class=xl6824191 width=131 style='border-left:none;width:98pt'>Mastermix/µL</td>
 
-
  <td class=xl6824191 width=87 style='border-left:none;width:65pt'>P176/µL</td>
 
-
  <td class=xl6824191 width=84 style='border-left:none;width:63pt'>P177/µL</td>
 
-
  <td class=xl6824191 width=90 style='border-left:none;width:68pt'>P178/µL</td>
 
-
  <td class=xl6824191 width=86 style='border-left:none;width:65pt'>P179/µL</td>
 
-
  <td class=xl6824191 width=86 style='border-left:none;width:65pt'>P180/µL</td>
 
-
  <td class=xl6824191 width=86 style='border-left:none;width:65pt'>P181/µL</td>
 
-
  <td class=xl6824191 width=86 style='border-left:none;width:65pt'>P182/µL</td>
 
-
  <td class=xl6824191 width=86 style='border-left:none;width:65pt'>P183/µL</td>
 
-
  <td class=xl6824191 width=86 style='border-left:none;width:65pt'>P184/µL</td>
 
-
</tr>
 
-
<tr height=29 style='mso-height-source:userset;height:21.75pt'>
 
-
  <td height=29 class=xl6624191 width=143 style='height:21.75pt;border-top:
 
-
  none;width:107pt'><span lang=EN-US style='mso-ansi-language:EN-US'>DNA</span></td>
+
[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]<br />
-
  <td class=xl6524191 style='border-top:none;border-left:none'>-</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>4,3</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>2,3</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>3,0</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>2,4</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>6,4</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>5,0</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>4,2</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>4,0</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>4,0</td>
+
-
</tr>
+
<p style="font-size:13px; color:#003399;"><b>Comments</b>: Sequence analysis of pCerulean_Vp1up.</p>  
-
<tr height=29 style='mso-height-source:userset;height:21.75pt;mso-yfti-irow:
+
-
 
+
-
  1'>  
+
-
  <td height=29 class=xl6624191 width=143 style='height:21.75pt;border-top:
+
-
 
+
-
  none;width:107pt'><span lang=EN-US style='mso-ansi-language:EN-US'>BSA (10x)</span></td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>10</td>
+
-
  <td rowspan=4 class=xl6524191 style='border-top:none'>3</td>
+
-
  <td rowspan=4 class=xl6524191 style='border-top:none'>3</td>
+
-
  <td rowspan=4 class=xl6524191 style='border-top:none'>3</td>
+
-
  <td rowspan=4 class=xl6524191 style='border-top:none'>3</td>
+
-
  <td rowspan=4 class=xl6524191 style='border-top:none'>3</td>
+
-
  <td rowspan=4 class=xl6524191 style='border-top:none'>3</td>
+
-
  <td rowspan=4 class=xl6524191 style='border-top:none'>3</td>
+
-
  <td rowspan=4 class=xl6524191 style='border-top:none'>3</td>
+
-
  <td rowspan=4 class=xl6524191 style='border-top:none'>3</td>
+
-
</tr>
+
-
<tr height=29 style='mso-height-source:userset;height:21.75pt;mso-yfti-irow:
+
-
 
+
-
  2'>
+
-
  <td height=29 class=xl6624191 width=143 style='height:21.75pt;border-top:
+
-
 
+
-
  none;width:107pt'><span lang=EN-US style='mso-ansi-language:EN-US'>Buffer
+
-
  4<span style='mso-spacerun:yes'>  </span>(10x)</span></td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>10</td>
+
-
</tr>
+
-
<tr height=29 style='mso-height-source:userset;height:21.75pt;mso-yfti-irow:
+
-
 
+
-
  3'>
+
-
  <td height=29 class=xl6624191 width=143 style='height:21.75pt;border-top:
+
-
 
+
-
  none;width:107pt'><span lang=EN-US style='mso-ansi-language:EN-US'>Enzyme
+
-
  XbaI<span style='mso-spacerun:yes'> </span></span></td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>5</td>
+
-
</tr>
+
-
<tr height=29 style='mso-height-source:userset;height:21.75pt;mso-yfti-irow:
+
-
 
+
-
  4'>
+
-
  <td height=29 class=xl6624191 width=143 style='height:21.75pt;border-top:
+
-
 
+
-
  none;width:107pt'><span lang=EN-US style='mso-ansi-language:EN-US'>Enzyme
+
-
  AgeI</span></td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>5</td>
+
-
</tr>
+
-
<tr height=29 style='mso-height-source:userset;height:21.75pt;mso-yfti-irow:
+
-
 
+
-
  5;mso-yfti-lastrow:yes'>
+
-
  <td height=29 class=xl6624191 width=143 style='height:21.75pt;border-top:
+
-
 
+
-
  none;width:107pt'><span lang=EN-US style='mso-ansi-language:EN-US'>H2O</span></td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>-</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>2,7</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>4,7</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>4,0</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>4,6</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>0,6</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>2,0</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>2,8</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>3,0</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>3,0</td>
+
-
</tr>
+
-
<tr height=29 style='mso-height-source:userset;height:21.75pt'>
+
-
  <td height=29 class=xl6624191 width=143 style='height:21.75pt;border-top:
+
-
 
+
-
  none;width:107pt'>Total volume</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>30</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>10</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>10</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>10</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>10</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>10</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>10</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>10</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>10</td>
+
-
  <td class=xl6524191 style='border-top:none;border-left:none'>10</td>
+
-
</tr> 
+
-
<tr height=0 style='display:none'>
+
-
  <td width=143 style='width:107pt'></td>
+
-
  <td width=131 style='width:98pt'></td>
+
-
  <td width=87 style='width:65pt'></td>
+
-
  <td width=84 style='width:63pt'></td>
+
-
  <td width=90 style='width:68pt'></td>
+
-
  <td width=86 style='width:65pt'></td>
+
-
</tr>
+
-
</table>
+
-
<br />
+
-
</ul>
+
<ul>
<ul>
-
<li>All 9 samples were loaded on a 1% agarose gel</li>
+
<li>Primer used: CMV-F</li>
-
<li>P176 = pSB1C3_hTERT clone1 </li>
+
<li>Plasmid sequenced: P303 </li>
-
<li>P177 = pSB1C3_hTERT clone2 </li>
+
<li>Sequence sample: ??</li>
-
<li>P178 = pSB1C3_betaglobin clone1</li>
+
<li>Stored in Geneious-folder: N-terminal Targeting --> pCerulean_Vp1up</li>
-
<li>P179 = pSB1C3_betaglobin clone2</li>
+
-
<li>P180 = pSB1C3_pTAV2(P5) clone1</li>
+
-
<li>P181 = pSB1C3_pTAV2(P5) clone2</li>
+
-
<li>P182 = pSB1C3_pAAV_RC(P5TATAless) clone1</li>
+
-
<li>P183 = pSB1C3_pAAV_RC(P5TATAless) clone2</li>
+
-
<li>P184 = pSB1C3_pAAV_RC(P5TATAless) clone3</li>
+
</ul>
</ul>
-
<br />
 
-
{| border="1" cellspacing="0" cellpadding="2" bordercolor="black"
 
-
!Sample
 
-
!Sample/µl]
 
-
!Loading dye (6x)/µl
 
-
!Expected size 1 (Geneious)
 
-
|--
 
-
|P176
 
-
|10 µl
 
-
|1,6 µl
 
-
|474 bp
 
-
|--
+
<gallery widths=600px heights=400px perrow=2 caption="pCerulean_Vp1up">
-
|P177
+
Image:Freiburg10 Seqanalysis pCerulean Vp1up 2010 27 08.jpg
-
|10 µl
+
</gallery>
-
|1,6 µl
+
-
|474 bp
+
-
 
+
-
|--
+
-
|P178
+
-
|10 µl
+
-
|1,6 µl
+
-
|509 bp
+
-
|--
+
-
|P179
+
-
|10 µl
+
-
|1,6 µl
+
-
|509 bp
+
-
|--
+
-
|P1780
+
-
|10 µl
+
-
|1,6 µl
+
-
|164 bp
+
-
|--
+
-
|P181
+
-
|10 µl
+
-
|1,6 µl
+
-
|164 bp
+
-
|--
+
-
|P182
+
-
|10 µl
+
-
|1,6 µl
+
-
|164 bp
+
-
|--
+
-
|P183
+
-
|10 µl
+
-
|1,6 µl
+
-
|164 bp
+
-
|--
+
-
|P184
+
-
|10 µl
+
-
|1,6 µl
+
-
|164 bp
+
-
 
+
-
|--
+
-
 
+
-
|}
+
-
{| align=right
+
-
|}
+
-
[[File:9 Proben tert, beta, p5.jpg|400px]]
+
<br />
<br />
 +
<b>Results:</b> <p style="color:#00bbff;">Results are good. Insertion of the Vp1up region can be confirmed.</p>
<br />
<br />
-
<p style="font-size:13px; color:#66bbff;"><b>Comments:</b> hTERT and betaglobin_mVenus is inserted in pSB1C3, pTAV(P5) and pAAV_RC (P5TATAless) will be sequenced</p><br>
+
<b>Next steps: Fuse pCerulean_Vp1up to the NLS. </b>  
-
Primer: Reverse Primer VR2
+
-
 
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Cell culture</b></p>====
+
-
Investigators: Adrian, Kerstin, Patrick
+
<br />
<br />
-
=====Transfection on HEK293=====
 
<br />
<br />
-
The motivation: first we want to create new stock of AAV without GOI, the next viral stock is with correct TK/GMK, the last stocks are with different YFP-amounts to check if the amount of GOI is a critical step for transduction effency (transgene expression).
+
 
 +
[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]<br />
 +
 
 +
====<p style="font-size:15px; background-color:#66bbff;"><b>Hybridisation and cloning of loop insertions</b></p>====
 +
<b>Investigator: Achim, Volker</b>
<br />
<br />
 +
<p style="font-size:13px;"><b>We hybridized the oligos for the different loop insertions and cloned them into the pSB1C3_BLA backbone.</b> </p>
 +
 +
<p style="font-size:13px; color:#cc0033;"><b>Hybridisation</b> </p>
 +
*4 Inserts contained overlapping ends and had to be filled up using Klenov fragments. We therefore added Klenow buffer and dNTPs to those samples.
{| border="1"
{| border="1"
-
| Plate  || align="right" |pAAV_RC/µg|| align="right" |pHelper/µg|| align="right" |GOI/µg
+
| align="left" | '''Components''' ||align="left"| '''453 BAP''' ||align="left"| '''587 BAP''' ||align="left"| '''587 KO BAP'''||align="left"| '''453 RGD'''||align="left"| '''587 RGD'''||align="left"| '''587 KO RGD'''||align="left"| '''453 HIS'''||align="left"| '''587 HIS'''||align="left"| '''587 KO HIS'''||align="left"| '''587 KO EMPTY'''||align="left"| '''453 Z34C'''||align="left"| '''587 Z34C'''||align="left"| '''587 KO Z34C'''||align="left"| '''587 KO Z34C SPACER'''
|-
|-
-
| 1|| align="right" |3,3 || align="right" | 3,3|| align="right" | no GOI
+
| align="left" | Oligo 1||align="left"|O124: 10 ||align="left"|O126: 10 ||align="left"|O128: 10 ||align="left"|O130: 10 ||align="left"|O132: 10 ||align="left"|O134: 10 ||align="left"|O135: 10 ||align="left"|O137: 10 ||align="left"|O139: 10 ||align="left"|O141: 10 ||align="left"|O143: 10 ||align="left"|O145: 10 ||align="left"|O147: 10 ||align="left"|O149: 10
|-
|-
-
| 2|| align="right" |6,6 || align="right" | 3,3|| align="right" | no GOI
+
| align="left" | Oligo 2 ||align="left"|O125: 10 ||align="left"|O127: 10 ||align="left"|O129: 10 ||align="left"|O131: 10 ||align="left"|O133: 10 ||align="left"|O151: 10 ||align="left"|O136: 10 ||align="left"|O138: 10 ||align="left"|O140: 10 ||align="left"|O142: 10 ||align="left"|O144: 10 ||align="left"|O146: 10 ||align="left"|O148: 10 ||align="left"|O150: 10
|-
|-
-
| 3|| align="right" |3,3 || align="right" | 3,3|| align="right" | 3,3 TK_GMK clone1
+
| align="left" | TrisHCl pH8||align="left"|4||align="left"|4||align="left"|4||align="left"|4||align="left"|4||align="left"|4 ||align="left"|4||align="left"|4||align="left"|4||align="left"|4||align="left"|-||align="left"|-||align="left"|-||align="left"|-
|-
|-
-
| 4|| align="right" |3,3 || align="right" | 3,3|| align="right" | 3,3 TK_GMK clone1
+
| align="left" | 5mM MgCl2||align="left"|8||align="left"|8||align="left"|8||align="left"|8||align="left"|8||align="left"|8 ||align="left"|8||align="left"|8||align="left"|8||align="left"|8||align="left"|-||align="left"|-||align="left"|-||align="left"|-
|-
|-
-
| 5|| align="right" |3,3 || align="right" | 3,3|| align="right" | 3,3 TK_GMK clone2
+
| align="left" | Klenow Buffer||align="left"|-||align="left"|-||align="left"|-||align="left"|-||align="left"|-||align="left"|- ||align="left"|-||align="left"|-||align="left"|-||align="left"|-||align="left"|4||align="left"|4||align="left"|4||align="left"|4
|-
|-
-
| 6|| align="right" |3,3 || align="right" | 3,3|| align="right" | 3,3 YFP
+
| align="left" | dNTP Mix||align="left"|-||align="left"|-||align="left"|-||align="left"|-||align="left"|-||align="left"|- ||align="left"|-||align="left"|-||align="left"|-||align="left"|-||align="left"|1||align="left"|1||align="left"|1||align="left"|1
|-
|-
-
| 7||  align="right" |3,3 || align="right" | 3,3|| align="right" | 10 YFP
+
| align="left" | H2O||align="left"|8||align="left"|8||align="left"|8||align="left"|8||align="left"|8||align="left"|8 ||align="left"|8||align="left"|8||align="left"|8||align="left"|8||align="left"|15||align="left"|15||align="left"|15||align="left"|15
-
|-
+
-
| 8|| align="right" |3,3 || align="right" | 3,3|| align="right" | 20 YFP
+
-
|-
+
-
| 9|| align="right" |3,3 || align="right" | 3,3|| align="right" | 40 YFP
+
-
|-
+
-
| 10|| align="right" |3,3 || align="right" | 3,3|| align="right" | 60 YFP
+
|-
|-
 +
| align="left" | <b>Total volume</b>||align="left"| <b>40</b> ||align="left"| <b>40</b> ||align="left"| <b>40</b> ||align="left"| <b>40</b> ||align="left"| <b>40</b> ||align="left"| <b>40</b> ||align="left"| <b>40</b> ||align="left"| <b>40</b> ||align="left"| <b>40</b> ||align="left"| <b>40</b> ||align="left"| <b>40</b> ||align="left"| <b>40</b> ||align="left"| <b>40</b> ||align="left"| <b>40</b>
|}
|}
-
=====Transduction of HT1080=====
+
*Hybridisation was carried out according to standard protocoll
-
<br />Motivation
+
*Klenov fill-in reaction:
-
The Motivation: checking survivalrate of Cells, with either no Virus, virus without GOI and virus with YFP (10µg stock)<br />
+
**Added 1 µl of NEB Klenow fragment to samples 11,12,13,14
-
The Plan: <br />
+
**incubated for 1h  @ 37°C
-
Plate I
+
<p style="font-size:13px; color:#cc0033;"><b>Digestion of pSB1C3_BLA vector and Samples 11-14 </b> </p>
 +
*We digested the standard vector with the 453 (Ssp/Sal) and the 587 (Bam/Pvu) standard. The samples that were filled in were also digested to create sticky ends.
 +
 
{| border="1"
{| border="1"
-
| control, no cells || align="right" |no GOI 150µl|| align="right" |10µg 500µl|
+
| align="left" | '''Components''' ||align="left"| '''V453''' ||align="left"| '''V587''' ||align="left"| '''11''' ||align="left"| '''12''' ||align="left"| '''13''' ||align="left"| '''14''' 
|-
|-
-
| control, no virus|| align="right" |no GOI 300µl|| align="right" | 10µg 750µl|
+
| align="left" | DNA ||align="left"| 2,5 ||align="left"| 2,5 ||align="left"| 3,5||align="left"| 3,5||align="left"| 3,5||align="left"| 3,5
|-
|-
-
|}
+
| align="left" | BSA (10x) ||align="left"| - ||align="left"| - ||align="left"| - ||align="left"| - ||align="left"| - ||align="left"| -
-
 
+
-
 
+
-
Plate II
+
-
{| border="1"
+
-
| control, no cells || align="right" |no GOI 150µl|| align="right" |10µg 500µl|
+
|-
|-
-
| control, no virus|| align="right" |no GOI 300µl|| align="right" | 10µg 750µl|
+
| align="left" | Buffer 4 (10x) ||align="left"| 2 ||align="left"| 2 ||align="left"| 2 ||align="left"| 2 ||align="left"| 2 ||align="left"| 2
|-
|-
-
|}
+
| align="left" | Enzyme 1||align="left"| Ssp: 1 ||align="left"| Bam: 1 ||align="left"| Ssp: 1 ||align="left"| Bam: 1 ||align="left"| Bam: 1 ||align="left"| Bam: 1 
-
 
+
-
Plate III
+
-
{| border="1"
+
-
| control, no cells || align="right" |no GOI 150µl|| align="right" |10µg 500µl|
+
|-
|-
-
| control, no virus|| align="right" |no virus|| align="right" | 10µg 750µl|
+
| align="left" | Enzyme 2||align="left"| Sal:1 ||align="left"| Pvu: 1 ||align="left"| Sal: 1 ||align="left"| Pvu: 1 ||align="left"| Pvu: 1 ||align="left"| Pvu: 1 
|-
|-
 +
| align="left" | H2O||align="left"| 13,5 ||align="left"| 13,5 ||align="left"| 12,5 ||align="left"| 12,5 ||align="left"| 12,5 ||align="left"| 12,5 
 +
|-
 +
| align="left" | <b>Total volume</b> ||align="left"| <b>20</b> ||align="left"| <b>20</b> ||align="left"| <b>20</b>||align="left"| <b>20</b>||align="left"| <b>20</b>||align="left"| <b>20</b>
|}
|}
 +
<p style="font-size:13px; color:#cc0033;"><b>Gel Extraction </b> </p>
-
=====Passaging and seeding of A431=====
+
[[Image:Freiburg10 27082010achim.JPG|500px]]
-
one 6er dish was prepared for making pictures and one T75 Flask.
+
<p style="font-size:13px; color:#cc0033;"><b>Ligation</b> </p>
-
<br />
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Cloning of pCerulean_VP1up_NLS</b></p>====
 +
<b>Investigator: Anissa </b>
 +
<p style="font-size:13px; color:red;"><b>Comment</b>:Cloning did not work, because no NLS-band could be seen in the gel... Will be repeated on monday in two new approaches. One time VP1up will be cloned into pSB1C3_NLS and recloned as a fusion-product into pCerulean. Another time the oligos of NLS will be hybridisized, cut with rescriction enzymes and purificated with the Qiaex II kit.Then the NLS will be ligated into the pCerulean_VP1up  </p>
 +
<br/>
 +
*Digestion:
-
====<p style="font-size:15px; background-color:#66bbff;"><b>BioBrick Assembly of pSB1C3_leftITR_CMV and pSB1C3_hGH_rightITR</b></p>====
 
-
Investigator: <b>Bea, Achim, Jo</B>
 
-
<br />
 
-
<p style="font-size:13px; color:#003399;"><b>Comments</b>: In order to proceed with the BioBrick assembly the pasmids pSB1C3_left ITR and pSB1C3_CMV were used and pSB1C3_rigthITR and pSB1C3_hGH were used in order to obatin plasmids with both sequences.</p>
 
-
 
-
<gallery widths=400px heights=400px perrow=2 caption="BioBrick assembly of pSB1C3_hGH_rightITR and pSB1C3_leftITR_CMV">
 
-
Image:Freiburg10 BioBrick assembbly of pSB1C3 leftITR CMV 09 08 2010.JPG|Cloning strategy of pSB1C3_leftITR CMV
 
-
Image:Freiburg10 BioBrick assembbly of pSB1C3 hGH rightITR 09 08 2010.jpg|pSB1C3_hGH_rightITR
 
-
</gallery>
 
-
 
-
<br />
 
-
<br />
 
-
<b>Digestion:</b> <br />
 
{| border="1"
{| border="1"
-
| '''components'''   || align="right" |'''pSB1C3_left ITR''' || align="right" |'''pSB1C3_CMV''' || align="right" |'''pSB1C3_right ITR''' || align="right" |''' pSB1C3_hGH'''
+
| components  || align="right" |Vector || align="right" |Insert
|-
|-
-
| DNA  ||  align="right" |13,4 ||  align="right" |9,5 ||  align="right" |13,6 ||  align="right" |6,6
+
| DNA  ||  align="right" |2,6 ||  align="right" | 32
|-
|-
-
| BSA (10x) ||  align="right" |2 ||  align="right" | 2 ||  align="right" |2 ||  align="right" | 2
+
| BSA (10x) ||  align="right" |1,5||  align="right" | -
|-
|-
-
| Buffer 4 (10x)||  align="right" |2 ||  align="right" |2 ||  align="right" |2 ||  align="right" | 2
+
| Buffer 4 (10x)||  align="right" |1,5 ||  align="right" | 4
|-
|-
-
|Enzyme1 || align="right" |1 (SpeI) ||  align="right" |1 (Xba1)||  align="right" |1 (EcoRI) ||  align="right" |1 (EcoRI)
+
|Enzyme ||  align="right" |1 PstI ||  align="right" | 2 PstI
|-
|-
-
|Enzyme2 || align="right" |1 (PstI)||  align="right" |1 (PstI) ||  align="right" |1 (XbaI)||  align="right" |1 (SpeI)
+
|Enzyme ||  align="right" |1 AgeI||  align="right" | 2 NgoMIV
|-
|-
-
|H2O||  align="right" |0,6 ||  align="right" |4,5 ||  align="right" |0,4 ||  align="right" |7,4
+
|H<sub>2</sub>O||  align="right" |7,4 ||  align="right" | -
|-
|-
-
|'''Total volume'''||  align="right" | 20||  align="right" |20 ||  align="right" | 20||  align="right" |20
+
|'''Total volume '''||  align="right" |15||  align="right" | 40
|}
|}
 +
<br>
 +
* Gel: that's only the picture of the 2% agarose gel for seperating the NLS. But no band could be seen.
 +
[[image:Freiburg10 NLS.jpg|400px ]]
 +
 +
====<p style="font-size:15px; background-color:#66bbff;"><b>DARPin E_01</b></p>====
 +
<b>Investigator: Bea </b>
 +
<br />
 +
<p style="font-size:13px; color:#66bbaa;"><b>General overview of DARPins:</b></p>
 +
Natural protein ankyrin repeat molecules are motifs which can be found in proteins. These motifs are mediating protein-protein interactions. This suggests that ankyrin repeat (AR) proteins can be used for designing binding molecules. In Kohl <i>et al.</i> (2003) and Binz et al. (2003) the authors designed a structural framework with fixed consensus regions and randomized positions of interacting residues.
<br />
<br />
-
Preparation of gel:<br />
+
The repetitive nature of the ankyrin proteins allows modifications in their variable and modular binding surface. Therefore consensus sequences of natural ankyrin proteins were used to design novel and stable scaffolds for binding proteins.
-
0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 115 Volt, running time: 45 minutes
+
Designed Ankyrin Proteins (DARPins) are well expressed, monomeric in solution, thermodynamically stable and have the ability to fold fast. In the publication of Steiner <i>et al.</i> (2008) screening libraries were created by useing the signal recognition particle (SRP) translocation pathway for phage display. The proteins containing the appropriate translocation signal sequence are efficiently displayed on filamentous phage particles. Screening for DARPins binding to specific target proteins like EGF-R (Erb1) were performed by phage ELISAs. The extracellular domain I-III of the receptor ErbB1 was fused to the Fc part of human IgG1 and used for selection. The previously described combinatorial N3C library was used as a template for SRP selection. After phage ELISA ans sequencing have been performed only one clone (E_01) could be selected with high-affinity binding characteristics. For broaden the diversity of ErbB1 DARPins three more clones were selected by epitope masking (E_67, E_68 and E_69). Binding and epitope localization experiments showed that all selected clones recognize a competing epitope as the dominant binder E_01, except of E_69 which recognizes an epitope not competing with E_01. The low diversity obtained for DARPins against ErbB1 suggests that the binding interfaces are well suited for dominant selection. Additionally the selected clones showed no cross-reactivity with other ErbB-family receptors (Figure 2C).
 +
<br/>
 +
<br/>
 +
<gallery widths=400px heights=400px>
 +
Image: Freiburg10 DARPin E 01 table Steiner et al.jpg
 +
</gallery>
<br />
<br />
 +
The dominant DARPpin E_01 has very high affinities to the target protein ErbB1 (Table 3) and can be used as a potential targeting molecule four our approach in fusing the DARPin to the N-terminal VP proteins.
 +
<br />
 +
<br/>
 +
<p style="font-size:13px; color:#66bbaa;"><b>Overview of DARPinE_01 used in our approach: </b></p>
 +
<gallery widths=600px heights=200px>
 +
Image:Freiburg10 DARPin E 01.jpg
 +
</gallery>
 +
The designed ankyrin repeat protein used as a potential targeting molecule consists of three internal capping repeats and the C-and N-terminal capping repeats. Each internal repeat module comprises of one beta-turn and two hydrophobic alpha-helices. The potential interaction residues are located in the beta-turn the first alpha-helices of the AR-proteins. The complete nucleotide and corresponding amino acid sequence can be found in the appendix.
 +
<gallery widths=400px heights=400px perrow=2>
 +
Image: Freiburg10 DARPin E 01 structure.jpg
 +
Image: Freiburg10 DARPin E 01 structure 2.jpg
 +
</gallery>
 +
<br/>
 +
[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]<br />
-
 
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Update VP1 insertion</b></p>====
-
[[Image: Freiburg10 9 8 2010 Digestion of psB1C3 rITR psB1C3 lITR psB1C3 CMV psB1C3 hGH125.jpg|200px]]
+
<b>Investigator: Anissa, Bea </b>
-
<p style="font-size:13px; color:#ff00ff;"><b>Oh, nice picture.. I can recognize many details :)*g* (Hanna) </b> </p>  
+
-
 
+
-
Expected sizes of constructs:
+
-
*pSB1C3_CMV:  650 bp
+
-
*pSB1C3_lITR: 2200 bp
+
-
*pSB1C3_hGH:  500 bp
+
-
*pSB1C3_rITR: 2200 bp
+
-
 
+
-
The correspnding bands were cut out and Gel-Extraction was performed according to protocol.
+
<br />
<br />
 +
<p style="font-size:13px; color:#cc0033;"><b>Done:</b> </p>
 +
<ul>
 +
<li> <b>pSB1C3_VP1up</b> was succesfully created by Achim. For details see labday from 2010-22-08.</li>
 +
<li> <b>pCerulean_VP1up</b> was suceesfully created by Anissa. pCerulean was sequenced and the VP1up which contains the upstream 137 aa´s of the VP1 protein could be successfully cloned into the plasmid backbone pCerulean (obtained by performeing a PCR).</li>
 +
<li><b>pSB1C3_NLS</b>Hybridization of the nuclear localization sequence (NLS) was successfully incorporated into the pSB1C3_CFP.  </li>
 +
<li>Today, Anissa tried to fuse the NLS to the VP1up sequence. Cutting the pSB1C3_NLS with NgomIV and PstI leads to a 45 nt fragment which normally can be resoluted in a 2% agarose gel. But no fragment could be detected in the gel (for further details see: Cloning of <b>pCerulean_VP1up_NLS</b>).
 +
<br />
 +
On monday another approach will be performed: The NLS oligos will be hybridized and digested with NgomIV and SpeI. With the Qiaex II Kit (protocol for desalting and concentrating DNA solutions),the hybrifized oligos will be purified. Parallel, another digestion approach will be conducted. </li>
 +
<gallery widths=300px heights=400px perrow=2 caption="Overview">
 +
Image:Freiburg10 Overview VP1 part1.jpg
 +
Image:Freiburg10 Overview VP1 part2.jpg
 +
</gallery>
 +
</ul>
 +
<p style="color:#00bbff;"> Next steps: </p>
 +
<ul>
 +
<li> We obtain pSB1C3_VP23_HSPG_ko by conducting a PCR with the pAAV_RC_final (which contains the integrated "cap". </li>
 +
<li> Fuse the obtained construct pSB1C3_Targeting Molcule to the pSB1C3_VP23_HSPG_ko. </li>
 +
<ul>
 +
<li>Affibody Z<sub>EGFR:1907</sub></li>
 +
<li>DARPin E_01</li>
 +
<li>6xHis Tag</li>
 +
<li>CFP</li>
 +
</ul>
 +
<li> Fuse pSB1C3_VP23_HSPG_ko_Targeting molecule to the construct pCerulean_VP1up_NLS.</li>
 +
<li> Finally, for providing the possibilty to express obtained construct in trans: perform a site-directed mutagenesis with the pAAV_RC_final to remove the startcodon of VP1.</li>
 +
</ul>
 +
[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]<br />
-
 
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Sequence analysis of pSB1C3_BLA_final (P309)</b></p>====
-
 
+
<b>Investigator: Bea </b>
-
 
+
-
 
+
<br />
<br />
 +
<p style="font-size:13px; color:#cc0033;"><I><b>GENERAL COMMENT:</b> pSB1C3_BLA_final (P309) was sent for sequencing. the beta lactamse was inserted and two site-directed mutagenesis have been performed in order to delete the loop insertion restriction enzymes in the CAT marker.</i> </p>
 +
<br />
 +
<p style="font-size:13px; color:#003399;"><b>Comments</b>: Sequence analysis of pSB1C3_BLA_final containing the two deleted restriction sites  (SspI and PvuII)in the CAT marker.</p>
 +
<ul>
 +
<li>Primer used: </li>
 +
<ul>
 +
<li>pQE-RP</li>
 +
<li>VF-2</li>
 +
</ul>
 +
<li>Plasmid sequenced: P309 </li>
 +
<li>Sequence sample: SB_9</li>
 +
<li>Stored in Geneious-folder: pSB1C3_BLA</li>
 +
</ul>
-
concentrations measured via NanoDrop:
+
<gallery widths=500px heights=400px perrow=2 caption="pSB1C3_BLA_final">
-
*pSB1C3_CMV: 13,51 ng/µl
+
Image:Freiburg10 Seqanalysis pSB1C3 BLA final P309 SspI.jpg
-
*pSB1C3_lITR: 18,49  ng/µl
+
Image:Freiburg10 Seqanalysis pSB1C3 BLA final P309 PvuII.jpg
-
*pSB1C3_hGH: 7,22  ng/µl
+
Image:Freiburg10 Seqanalysis pSB1C3 BLA final P309.jpg
-
*pSB1C3_rITR: 20,24  ng/µl
+
</gallery>
-
 
+
<br />
<br />
 +
<b>Results:</b> <p style="color:#00bbff;">The two performed site-directed mutagenesis performed at the backbone of the pSB1C3 containing the beta-lactamase can be confrimed as it can be seen in the two pictures above. Additionally the BLA sequence was sequenced aswell as the insertion of the BLA which can be confirmed with no mutations.</p>
-
<b>Ligation:</b> <br />
 
-
For ligation, T4 ligase was used.
 
<br />
<br />
-
* 1µl T4-Ligase buffer (10x)
+
<b>Next steps: This new plasmid can be used for further experiments. Clone the PCR prdouct of the pAAV_RC into the pSB1C3 and subclone the loop insertion motifs into the vector.</b>
-
* 8µl (Vector + Insert) mix
+
<br />
-
* 1µl T4-Ligase
+
<br />
-
Ligation was carried out as following:
+
-
* Ligation 1:
+
-
**pSB1C3_CMV:  4,4 ng/µl
+
-
**pSB1C3_lITR: 3,6  ng/µl
+
-
*Ligation 2:
+
-
**pSB1C3_hGH:  5,25 µl
+
-
**pSB1C3_rITR: 2,75 µl
+
 +
[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]<br />
-
<br /> Incubation for 40 minutes.
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-Preps and test digestion of pSB1C3_6xHis_middlelinker and pSB1C3_6xHis_mVenus</b></p>====
 +
<b>Investigator: Stefan</b><br>
 +
<ul>
 +
<b>Glycerol stocks were prepared:</b>
 +
<li>B258 = pSB1C3_6xHis_middlelinker clone1</li>
 +
<li>B259 = pSB1C3_6xHis_middlelinker clone2</li>
 +
<br />
-
<br />
+
<li>B260 = pSB1C3_6xHis_mVenus clone 1</li>
-
<b>Transformation:</b><br />
+
<li>B261 = pSB1C3_6xHis_mVenus clone 2</li>
-
Transformation carried out according to standard protocol (BL21). Cells were plated on an agar plate with chloramphenicol.
+
<br />
<br />
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Repetition: Cloning of SDM SspI and SDM PvuII</b></p>====
+
<b>Mini-Prep following the standard protocol</b>
-
Investigator: <b>Stefan</B><br />
+
<li>P310 = pSB1C3_6xHis_middlelinker clone1<br>
-
<p style="color:red;">Comments: Last week's cloning of SDM_SspI and SDM_PvuII didn't work out, so this is the repetition. In this approach the same restriction enzymes and amounts of DNA were used.</p> <br />
+
c= 185,31 ng/µl</li>
 +
<li>P311 = pSB1C3_6xHis_middlelinker clone2<br>
 +
c= 178,87 ng/µl</li>
<br />
<br />
-
*Vector: name: pSB1C3_SDM_SspI <b>P125</b>
 
-
*Insert: name: pSB1C3_SDM_PvuII <b>P129</b>
 
-
 
-
*buffer used: 4
 
-
*Restriction-enzymes used:
 
-
**BpmI (no. Lab: 144)
 
-
**ClaI (no.Lab: 152)
 
-
*DNA concentration (P125): 321,9 ng/µl
 
-
*DNA concentration (P129): 308,4 ng/µl
 
 +
<li>P312 = pSB1C3_6xHis_mVenus clone 1<br>
 +
c= 247,62 ng/µl</li>
 +
<li>P313 = pSB1C3_6xHis_mVenus clone 2<br>
 +
c= 268,98 ng/µl</li>
<br />
<br />
-
 
+
</ul>
-
<b>Digestion:</b> <br />
+
<b>Test digestion</b><br>
 +
<ul>
 +
<li>Restriction-enzymes used: EcoRI and PstI</li>
 +
</ul><br />
{| border="1"
{| border="1"
-
| '''components'''   || align="right" |'''volume of pSB1C3_SDM_SspI /µl''' || align="right" |'''volume of pSB1C3_SDm_PvuII /µl'''  
+
| align="left" | '''Components''' ||align="left"| '''P310+311''' ||align="left"| '''P312+313'''  
|-
|-
-
| DNA || align="right" |4,66 || align="right" |4,87
+
| align="left" | DNA ||align="left"| 2 ||align="left"| 2
|-
|-
-
| BSA (10x) || align="right" |2 || align="right" | 2
+
| align="left" | BSA (10x) ||align="left"| 1 ||align="left"| 1
|-
|-
-
| Buffer 4 (10x)|| align="right" |2 || align="right" |2
+
| align="left" | Buffer 4 (10x) ||align="left"| 1 ||align="left"| 1
|-
|-
-
|BpmI (no.Lab:144)|| align="right" |1 || align="right" |1
+
| align="left" | EcoRI ||align="left"| 0,5 ||align="left"| 0,5
|-
|-
-
|ClaI (no.Lab:152)|| align="right" |1 || align="right" |1
+
| align="left" | PstI ||align="left"| 0,5 ||align="left"| 0,5
|-
|-
-
|H2O|| align="right" |9,34 || align="right" |9,13
+
| align="left" | H<sub>2</sub>O ||align="left"| 5 ||align="left"| 5
|-
|-
-
|'''Total volume'''|| align="right" | 20|| align="right" |20
+
| align="left" | <b>Total volume</b> ||align="left"| <b>10</b> ||align="left"| <b>10</b>
|}
|}
<br />
<br />
-
<b>Gel extraction:</b> <br />
+
Incubation time: 90 minutes; Incubation temperature: 37°
-
Preparation of gel:<br />
+
-
0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 115 Volt, running time: 45 minutes
+
<br />
<br />
-
 
+
1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , running time: 55 minutes at 120 Volt <br />
-
Expected sizes of constructs:
+
-
*pSB1C3_SDM_SspI: 1819 bp
+
-
*pSB1C3_SDM_PvuII: 981 bp
+
-
 
+
-
The correspnding bands were cut out and Gel-Extraction was performed according to protocol.
+
<br />
<br />
-
 
-
[[File:Freiburg10 pSB1C3 SDM SspI and PvuII 090810.png|500px|left|thumb|]]<br />
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
<br />
<br />
-
 
+
[[File:Freiburg10 test digestion His middle+His mVenus.jpg|500px]]
-
concentrations measured via NanoDrop:
+
-
* Sspl: 19,85 ng/µl
+
-
* PvuII: 10,57 ng/µl
+
<br />
<br />
-
 
-
<b>Ligation:</b> <br />
 
-
For ligation, T4 ligase was used.
 
-
<br />
 
-
* 1µl T4-Ligase buffer (10x)
 
-
* 8µl (Vector + Insert) mix
 
-
* 1µl T4-Ligase
 
-
The Ligation was performed as following:
 
-
* SDM_SspI Volume: 4,03 µl
 
-
* SDM_PvuII Volume: 3,97 µl
 
<br />
<br />
-
* 1µl T4-Ligase buffer (10x)
+
<p style="font-size:13px; color:#003399;"><b>Comment</b>: Test digestion looks good. Clones 1 of each plasmid (P310 and p312) were sent in for sequencing. </p>
-
* 8µl (Vector + Insert) mix
+
-
* 1µl T4-Ligase
+
-
<br /> Incubating for 45 minutes.
+
 +
====<p style="font-size:15px; background-color:#66bbff;"><b>Repetition of test digestion of pAAV_RC_ins-rep-cap, pAAV_RC_ins-rep, pSB1C3_Rep40_RC_Insert and pSB1C3_Rep68_RC_Insert.</b></p>====
 +
<b>Investigator: Chris L.</b><br>
 +
<ul>
 +
<li>P250 = pAAV_RC_ins-rep clone1<br>c=467,50 ng/µl</li>
 +
<li>P314 = pAAV_RC_ins-rep-cap clone1<br>c=517,04 ng/µl</li>
 +
<li>P315 = pAAV_RC_ins-rep-cap clone2<br>c=444,33 ng/µl</li>
<br />
<br />
-
<b>Transformation:</b><br />
+
<li>P316 = pSB1C3_Rep40_ins clone 1<br>c=302,22 ng/µl</li>
-
Transformation performed according to the standard protocol (BL21). The cells were plated on a agar plate with chloramphenicol.
+
<li>P317 = pSB1C3_Rep40_ins clone 2<br>c=274,37 ng/µl</li>
-
<br />
+
<li>P318 = pSB1C3_Rep68_ins clone 1<br>c=491,69 ng/µl</li>
-
 
+
<li>P319 = pSB1C3_Rep68_ins clone 2<br>c=498,71 ng/µl</li>
-
====<p style="font-size:15px; background-color:#ff00ff;"><b>Design and ordering of primer for P40 Promoter BioBrick production</b></p>====
+
-
Investigator: <b>Hanna</B><br />
+
-
<br/>
+
-
In order to generate a P40 promoter BioBrick = "Cap-Promoter", oligos were designed and ordered at Sigma Aldrich:<br/>
+
-
[[File:Freiburg10 P40.jpg|450px]]
+
-
<br/>
+
-
 
+
-
=== 85. labday 10.08.2010===
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Retrafo with RepCap gene (Mr.Gene)</b></p>====
+
-
<b>Investigator: Bea</b><br>
+
-
<p style="font-size:13px; color:#66bbff;"><b>Comments</b>: We received the rep_cap insert ordered from Mr. Gene today. In order to used and clone the insert into the desired sequences a retrafo has to be performed.  </p>
+
-
<b> Re-transformation: </b>
+
-
<ul>
+
-
<li>Construct used: pMA_REP_CAP</li>
+
-
<li> We reveived 5 µg (lyophilzied). These were resuspended in 100 µL H<sub>2</sub>O and vortexted.</li>
+
-
<li>c = 50 ng/µL</li>
+
-
<li>c = Plasmid need to be diluted in order to obatain the final 500pg</li>
+
-
<li>Dilution: 1:100</li>
+
-
<li>100 µL aliquot of <b>XL1-B</b> were thawed on ice. The proper amount of 1µL was added to the cells.</li>
+
-
<li>Transformed cells were plated on LB plates containing ampicillin</li>
+
</ul>
</ul>
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Continuation: Cloning Rep40 & Rep52 into pSB1C3</b></p>====
 
-
A midi prep of the two Rep52+DMSO clones was performed yielding concentrations of 261,35 ng/µl (clone 1) and 240,47 ng/µl (clone 2). Unfortunately we noticed today that we should not use EcoRI to clone Rep52 into pSB1C3 because EcoRI cuts two times within the sequence of Rep52.
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Midi-Prep of pAAV_RC, pAAV_RC_1.2 SDM SalI (P158), pAAV_RFC25_mGMK_TK30 clone 1 (P81) and 2 (P82)</b></p>====
 
-
Investigators: Patrick, Chris <br>
 
-
 
-
The Midi-Preps were performed according to the standard protocol yielding the following concentrations:
 
-
 
-
{| border="1"
 
-
| pAAV_RC || align="right" |P81a|| align="right" |P81b|| align="right" |P82a|| align="right" |P82b|| align="right" |P158a|| align="right" |P158b
 
-
|-
 
-
| 1348,49 ng/µl||  align="right" |1783,86 ng/µl||  align="right" | 2096,25 ng/µl|| align="right" |1915 ng/µl|| align="right" |1469,35 ng/µl|| align="right" |1834,25 ng/µl|| align="right" |1771,27 ng/µl
 
-
|-
 
-
|}
 
<br>
<br>
-
The annotations a and b were added because two midi-preps of P81, P82 and P158 were carried out.
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Results of sequencing pSB1C3_pTAV2(P5) and Psb1C3_pAAV_RC(P5TATAless)</b></p>====
+
<b>Test digestion:</b>
-
<b>Investigator: Jessica</b><br>
+
-
 
+
-
[[image:mistake.png|thumb|right]]
+
-
@Jessi: Which clones are ok, which not?? text does not correspond to pictures! (Bea)
+
-
<br />
+
-
[[File:Freiburg10_Sequence_alignment_P5 mit TATA clone1neu.jpg|500px|left|thumb|]]
+
<br>
<br>
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
sequencing of '''P180''' was successful, P5 with TATAbox is in pSB1C3
 
-
[[File:Freiburg10_Sequence_alignment_P5TATAless clone2neu.jpg|500px|left|thumb|]]
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
sequencing of '''P183''' was successful but there is a G missing --> plamid is thrown away
 
-
[[File:Freiburg10_Sequence_alignment_P5TATAless clone3.jpg|500px|left|thumb|]]
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
sequencing of '''P184''' was successful, P5 is in without TATAbox. this plasmid can be used. <br>
 
-
<br>
 
-
'''P181''' and '''P180''' are thrown away because of the result of the test digestion.
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Repetition: BioBrick production of Rep40 and Rep 52</b></p>====
 
-
Investigator: <b>Anna</B><br />
 
-
 
-
<p style="font-size:13px; color:#68bbff;">'''''Comments''''': PCR was repeated once again (that was bad!), because digestion was done with the wrong enzymes (EcoRI HF and SpeI). There are some differences in the PCR programm in comparison to the last (see 09.10.)</p> <br>
 
-
<br>
 
-
'''PCR
 
-
 
{| border="1"
{| border="1"
-
| '''Ingredients''' || align="right" |'''Rep 40''' ||align="right"|'''Rep52'''
+
| components ||volume of '''P250'''/µl ||volume of '''P314'''/µl ||volume of '''P315'''/µl ||volume of '''P316'''/µl ||volume of '''P317'''/µl ||volume of '''P318'''/µl ||volume of '''P319'''/µl
|-
|-
-
| 5X Phusion HF buffer || align="right" |10 µl||align="right"|10 µl
+
| DNA  || 1 || 1 || 1 || 1 || 1 || 1 || 1
|-
|-
-
| 10 mM dNTP mix|| align="right" |1µl ||align="right"|1 µl
+
| BSA (10x) ||1 ||1 || 1 || 1 || 1 || 1 ||1  
|-
|-
-
| forward primer: O094 || align="right" |2,5µl||align="right"|2,5 µl
+
| Buffer 2 (10x) ||1 ||1 || 1 || 1 || 1 || 1 || 1
|-
|-
-
| reverse primer: O0096/ O097 || align="right" |2,5 µl||align="right"|2,5 µl
+
|Enzyme Acc65I ||0,75 ||0,75 ||0,75 || - || - || - || -
|-
|-
-
| DNA Template 1:100 (1ng)|| align="right" |1 µl||align="right"| 1 µl
+
|Enzyme XcmI ||0,5 ||0,5 ||0,5 || - || - || - || -
|-
|-
-
| DMSO || align="right" |1 µl||align="right"|1 µl
+
|Enzyme HindIII || - || - || - ||0,5 ||0,5 ||0,5 ||0,5
|-
|-
-
| Phusion Polymerase|| align="right" |0,5 µl||align="right"| 0,5 µl
+
|Enzyme BstEII || - || - || - ||0,5 ||0,5 ||0,5 ||0,5
 +
|-
 +
|H2O ||5,75 ||5,75 ||5,75 || 6 || 6 || 6 || 6
|-
|-
-
|H<sub>2</sub>O|| align="right" |31,5 µl||align="right"|31,5 µl
+
|'''Total volume /µl'''||10 ||10 ||10 ||10 ||10 ||10 ||10
-
|-
+
-
|Total volume|| align="right" |50 µl||align="right"| 50 µl
+
|}
|}
-
<br>
 
-
 
-
'''PCR programm
 
-
 
-
{| border="1"
 
-
|'''PCR Program'''|| align="right" |'''temperature/ °C'''|| align="right" |'''Time''' || align="right" |'''PCR products
 
-
|-
 
-
|1||  align="right" |98 ||  align="right" |1min||  align="right" |
 
-
|-
 
-
|2||  align="right" |98 ||  align="right" |15s||  align="right" |
 
-
|-
 
-
|8x||  align="right" |*** ||  align="right" |15s||  align="right" |'''62,5°C
 
-
|-
 
-
|3 ||  align="right" |72||  align="right" |***||  align="right" |'''25s 
 
-
|-
 
-
|4 ||  align="right" |98||  align="right" | 15s||  align="right" |
 
-
|-
 
-
|19x||  align="right" |***||  align="right" |15s||  align="right" |'''67°C
 
-
|-
 
-
|5||  align="right" |72||  align="right" |***||  align="right" |'''25s
 
-
|-
 
-
|6x||  align="right" |72||  align="right" |5min||  align="right" | 
 
-
|-
 
-
|Hold||  align="right" |4||  align="right" |  ||  align="right" |
 
-
|}
 
-
<br>
 
-
 
-
'''Agarose gel
 
-
 
-
0.5  g Agarose, 50 mL TAE (1 %),3 µL GELRED, at 120 Volt, running time: 45 minutes
 
<br />
<br />
 +
Incubation time: 1 h, Incubation temperature: 37° for P250, P314 and P315
 +
<br />
 +
Incubation time: 45 min, Incubation temperature: 37° with HindIII for P316, P317, P318 and P319 ; 45 min, Incubation temperature: 60° with BstEII for P316, P317, P318 and P319
 +
>br />
 +
Preparation of gel:<br />
 +
1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 120 Volt, running time: 45 minutes
<br />
<br />
-
{| border="1" cellspacing="0" cellpadding="2" bordercolor="black"
 
-
!Sample
 
-
!Sample / µl]
 
-
!Loading dye (6x) / µl
 
-
!Expected size
 
-
 
-
|--
 
-
|p22 (Rep40)
 
-
|50 µl
 
-
|8 µl
 
-
|940 bp
 
-
|--
 
-
|p23 (Rep52)
 
-
|50 µl
 
-
|8 µl
 
-
|~1200 bp
 
-
|--
 
-
|}
 
{| align=right
{| align=right
|}
|}
Line 3,149: Line 1,354:
{| border="1"
{| border="1"
|
|
-
!Marker /µL
+
!Marker
-
!Sample Rep40 /µl
+
!Sample P250 10 /µl
-
!Sample Rep52 /µl
+
!Sample P314 10 /µl
 +
!Sample P315 10 /µl
 +
!Sample P316 10 /µl
 +
!Sample P317 10 /µl
 +
!Sample P318 10 /µl
 +
!Sample P319 10 /µl
|-
|-
!Lane  
!Lane  
 +
|1
 +
|3
 +
|4
 +
|5
|7
|7
-
|58
+
|8
-
|58
+
|9
 +
|10
|-
|-
|}
|}
<br />
<br />
-
 
+
[[File:Freiburg10 pAAV RC ins-rep-cap pSB1C3 Rep40 ins pSB1C3 Rep68 ins.png]]
-
[[File:Freiburg10_Rep40_52.jpg|500px|left|thumb|]]
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
<br />
<br />
 +
<p style="font-size:13px; color:#003399;"><b>Comment</b>: The P314 and P315 looks like the control vector. The RepCap Vector_SDM_InsPvuII insertion didn´t work. The P316, P317, P318 and P318 was cut with the "wrong" enzymes. The same fragment size as the original vector appeared. Next time cut with EcoRI. </p>
-
<p style="font-size:13px; color:#68bbff;">'''''Comments''''': Digestion of the samples will be done tomorrow with XbaI and SpeI.</p> <br>
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Midi-Prep of pSB1C3_VCK_Bla</b></p>====
-
<br />
+
-
'''Gelextraction
+
'''Investigators: Chris W. <br>
 +
<p style="font-size:13px; color:#003399;"><b>Comment</b>: Midi-Preps of P 320, B257</p>
 +
The Midi-Preps were performed according to the standard protocol yielding the following concentrations:
-
Elution was done with 20 µl Pe buffer following the standard protocol.<br />
+
{| border="1"
 +
| plasmid-no. || align="right" |P320
 +
|-
 +
| concentration (ng/µl)|| align="right" |408
 +
|-
 +
|}
 +
<br>
-
c(Rep40)= 23,1 ng/µl<br />
+
===103. labday 28.08.2010===
-
c(Rep52)= 78,0 ng/µl
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-Prep of pSB1C3_NLS</b></p>====
-
 
+
<b>Investigator: Stefan</b><br>
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Preparation of competent XL1blue and testtrafo</b></p>====
+
comments: No glycerol stock and test digestion was performed because bacteria culture inocculated from glyerol stock (B233).
-
<b>Investigator: Jessica</b><br>
+
<ul>
-
preparation of competent E.coli XL1blue was made according to the standardprotocol (91 eppis with '''60µl''') they are in the green boxes in -80°C<br>
+
<b>Mini-Prep following the standard protocol</b>
-
testtrafo was made
+
<li>P321 = pSB1C3_NLS (1)<br>
-
<ul><li>one plate with 50pg/µl</li>
+
c= 180,45 ng/µl</li>
-
<li>one plate with 100pg/µl</li>
+
<li>P322 = pSB1C3_NLS (2)<br>
 +
c= 187,26 ng/µl</li>
 +
<br />
</ul>
</ul>
-
...trafo is failed because amp was missing on the plates (resistence in pUC). trafo will be repeated on aug 11th by Tobi...
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Repetition: Mini-Prep and test digestion of pSB1C3_SDM_SspI/PvuII</b></p>====
+
<p style="font-size:13px; color:#cc0033;"><I><b>COMMENT (Patrick):</b> These mini-preps will be thrown away because B233 was used for inoculation due to a wrong entry in our plasmid excel sheet.  B233 this is NOT the sequenced clone ! We sequenced B234/P282. Therefore 2x10 ml DYT + Cm were inoculated with B234. </i> </p>
-
'''Investigator: Adrian, Stefan'''<br>
+
 
-
<br>
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Cell culture</b></p>====
-
<p style="font-size:18px; font-weight: bold; color:#66bbff;"><u>Plasmid Mini-Prep</u></p>
+
=====Harvest viral Stock and Transductiot of HT and A431 cells=====
-
*vector name: pSB1C3_SDM_SspI/PvuII
+
<ol>
 +
<li>P169: 100.000 cells</li>
 +
<li>P169: 200.000 cells</li>
 +
<li>P169: 300.000 cells</li>
 +
<li>P169: 400.000 cells</li>
 +
<li>P169: 500.000 cells</li>
 +
<li>P169: 600.000 cells</li>
 +
<li>P169: 700.000 cells</li>
 +
<li>P262: 100.000 cells</li>
 +
<li>P262: 200.000 cells</li>
 +
<li>P262: 300.000 cells</li>
 +
<li>P262: 400.000 cells</li>
 +
<li>P262: 500.000 cells</li>
 +
<li>P262: 600.000 cells</li>
 +
<li>P262: 700.000 cells</li>
 +
</ol>
<br />
<br />
-
<u>Glycerol Stocks</u> <br>
+
200.000 cells per well<br />
-
pSB1C3_SDM_SspI/PvuII
+
<table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
-
Only clone 1 grew, therefore there is just one stock = B159
+
<tr>
 +
<th width="80"> </th>
 +
<th width="80">1</th>
 +
<th width="80">2</th>
 +
<th width="80">3</th>
 +
</tr>
 +
<tr>
 +
<td>A</td>
 +
<td>control, no cells</td>
 +
<td>1</td>
 +
<td>2</td>
 +
</tr>
 +
<tr>
 +
<td>B</td>
 +
<td>control, no virus</td>
 +
<td>1</td>
 +
<td>2</td>
 +
</tr>
 +
</table>
 +
<table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 +
<tr>
 +
<th width="80"> </th>
 +
<th width="80">1</th>
 +
<th width="80">2</th>
 +
<th width="80">3</th>
 +
</tr>
 +
<tr>
 +
<td>A</td>
 +
<td>control, no cells</td>
 +
<td>3</td>
 +
<td>4</td>
 +
</tr>
 +
<tr>
 +
<td>B</td>
 +
<td>control, no virus</td>
 +
<td>3</td>
 +
<td>4</td>
 +
</tr>
 +
</table>
 +
<table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 +
<tr>
 +
<th width="80"> </th>
 +
<th width="80">1</th>
 +
<th width="80">2</th>
 +
<th width="80">3</th>
 +
</tr>
 +
<tr>
 +
<td>A</td>
 +
<td>control, no cells</td>
 +
<td>5</td>
 +
<td>6</td>
 +
</tr>
 +
<tr>
 +
<td>B</td>
 +
<td>control, no virus</td>
 +
<td>5</td>
 +
<td>6</td>
 +
</tr>
 +
</table>
-
<p style="font-size:15px; font-weight: bold; color:#66bbff;">Test digestion</p>
+
<table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
-
*buffer used: 2
+
<tr>
-
*Restriction-enzymes used:
+
<th width="80"> </th>
-
**Enzyme 1 SspI
+
<th width="80">1</th>
-
**Enzyme 2 PvuII
+
<th width="80">2</th>
-
*Plasmid: pSB1C3_SDM_SspI/PvuII:
+
<th width="80">3</th>
-
**Plasmid-Number: P185; DNA concentration: 47,9 ng/µL <br />
+
</tr>
-
 
+
<tr>
-
<p style="font-size:13px; color:#ff0000;">comment: DNA was eluted into previously used tube, therefore contamination is possible.</p> <br>
+
<td>A</td>
 +
<td>control, no cells</td>
 +
<td>7</td>
 +
<td>8</td>
 +
</tr>
 +
<tr>
 +
<td>B</td>
 +
<td>control, no virus</td>
 +
<td>7</td>
 +
<td>9</td>
 +
</tr>
 +
</table>
 +
<table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 +
<tr>
 +
<th width="80"> </th>
 +
<th width="80">1</th>
 +
<th width="80">2</th>
 +
<th width="80">3</th>
 +
</tr>
 +
<tr>
 +
<td>A</td>
 +
<td>control, no cells</td>
 +
<td>11</td>
 +
<td>13</td>
 +
</tr>
 +
<tr>
 +
<td>B</td>
 +
<td>10</td>
 +
<td>12</td>
 +
<td>14</td>
 +
</tr>
 +
</table>
 +
=====Seeding AAV293 for Transfection at 30.8=====
<br />
<br />
-
 
+
8 plates with 200.000 cells each were seeded
-
{| border="1"
+
-
| align="left" | '''Components''' ||align="left"| <b>P185</b> Volume/µL ||align="left"| <b>P51.1</b> Volume/µL
+
-
|-
+
-
| align="left" | DNA ||align="left"| 18,1 ||align="left"| 6,8  
+
-
|-
+
-
| align="left" | BSA (10x) ||align="left"| 2,5 ||align="left"| 2,5
+
-
|-
+
-
| align="left" | Buffer no. 2 (10x) ||align="left"| 2,5 ||align="left"| 2,5
+
-
|-
+
-
| align="left" | Enzyme 1 SspI ||align="left"| 0.5 ||align="left"| 0.5
+
-
|-
+
-
| align="left" | Enzyme 2 PvuII ||align="left"| 0.5 ||align="left"| 0.5
+
-
|-
+
-
| align="left" | H<sub>2</sub>O ||align="left"| 0,9 ||align="left"| 12,2
+
-
|-
+
-
| align="left" | '''Total volume''' ||align="left"| <b>10</b>  ||align="left"| <b>10</b> |
+
-
|}
+
<br />
<br />
 +
====<p style="font-size:15px; background-color:#66bbff;"><b>Picking clones from the ViralBricks</b></p>====
 +
Investigator: Volker
-
*Incubation: 45 min
+
Clones from all 14 transformations of the ViralBricks were picked. Because the probability that the ligation worked and that the synthesized oligos do not conatin mutations is quite bad I picked 8 to 10 clones from each ligation, resulting in approximately 150 clones at all. The clones were used to inoculate 5ml of a mixture of 60%LB-media nd 40%DYP-media because our stocks were not sufficient for this experiment.
-
<br />
+
-
<p style="font-size:15px; font-weight: bold; color:#66bbff;">Agarose-Gel:</p>
+
-
<br />
+
-
0.4 g Agarose, 50 mL <b>TAE</b>, 3 µL GELRED, at 115 Volt, running time: 45 minutes
+
-
<br />
+
-
{| align=right
+
===104. labday 29.08.2010===
-
|}
+
====<p style="font-size:15px; background-color:#66bbff;"><b>P282/B234 mini-prep</b></p>====
-
<br />
+
... was performed according to the standard protocol. <br>
-
*Marker: GeneRuler ladder mix
+
Labelling:
-
{| border="1"
+
* P323, pSB1C3_NLS clone 2.1, 129 ng/µl
-
|
+
* P324, pSB1C3_NLS clone 2.2, 141 ng/µl
-
!Marker /µL
+
Investigator: Patrick
-
!Sample P185 /µL
+
-
!Sample P51.1 /µL
+
-
|-
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Digestion of pSB1C3_NLS (P324)</b></p>====
-
!Lane
+
-
|7
+
-
|17
+
-
|17
+
-
|-
+
-
|}
+
-
<br />
+
-
One cut is expected (PvuII in CFP).<br>
+
The NLS was cut out with NgoMIV and EcoRI, therefore the expected size of the hoped-for fragment is 55 bp. About 30 ng DNA can be detected in a 1% gel. According to my calculation there will be maximal 80 ng insert. <br>
-
[[File:Freiburg10 SspI PvuII test digestion.png|300px]] <br>
+
55bp/2019bp = 0,027 <br>
-
<br>
+
0,027x140 ng/µl = 3,81 ng/µl <br>
-
<b>Comment: There are 6 bands now. This test digestion again looks very different from that of P157.2 done by Jessica (lab day 82). Results should be discussed tomorrow.</b><br>
+
80 ng/3,81 ng/µl = 21 µl <br>
<br>
<br>
 +
Digestion: 21 µl pSB1C3_NLS (P324), 2,5 µl EcoRI, 2,5 µl NgoMIV, 3 µl Buffer 4, 1 µl H2O. <br>
 +
Digestion Time: 2 h 20 minutes.
-
<br/>
+
The 2% agarose gel was loaded with 36 µl digesteion product including 6 µl 30% glycerol solution. I did not use loading dye because i to prevent that the dye overlay the mutual 55 bp insert.
-
<br/>
+
 +
[[File:Pat2908.JPG|500px]]
-
====<p style="font-size:15px; background-color:#66bbFF;"><b>Picking clones of pSB1C3_SDM_SspI/PvuII</b></p>====
+
The gelextraction was performed according to the the standard protocl. The resolution of the picture ist not sufficient to be able to see the band clearly as the gel was put onto the UV-light table a very thin band with the expected size could be seen. <br>
-
<p><b>Investigator: Stefan</b></p>
+
DNA concentration measurement:
-
<p style="font-size:13px; color:#66bbff;"><b>Comments:</b> Test digestion didn't work out, three additional clones were picked from the plate we get from yesterday's Trafo to retry tomorrow.</p>
+
3,28 & 7,7 ng/µl (two measurements). Especially the second value is doubtable because according to my calculation above the insert conccentration cant be higher than 3,81 ng/µl. Labeled: NLS, put into 4°C room.
-
<ul>
+
-
<li>Bacterial strain used: BL21</li>
+
-
<li>Inoculating of 10 mL DYT medium containing 10µL chloramphenicol</li>
+
-
<li> Put in 37°C room on shaker at 20.45hours.</li>
+
-
</ul><br><br>
+
-
=== 86. labday 11.08.2010===
+
New digestion over night following a gelrun tomorrow: 23 µl pSB1C3_NLS (P324), 2 µl EcoRI, 2 µl NgoMIV, 3 µl Buffer 4. <br>
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Biobrick Assembly:Picking Clones, Miniprep, Testdigestion of CMV&lITR/ hGH & rITR</b></p>====
+
-
Investigator: <b>Chris L., Achim</B>
+
-
Test Digestion showed that the cloning was succesful.
+
-
[[Image:Freiburg10_assembly1.jpg]]
+
-
<br />
+
-
<br />
+
-
NanoDrop concentrations:
+
-
*hGH_rITR1:105,15 ng/µl
+
-
*hGH_rITR2:96.97 ng/µl
+
-
*CMV_lITR1:231.61 ng/µl
+
-
*CMV_lITR2:129.19 ng/µl
+
-
<br />
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Biobrick Assembly: bSB1C3_lITR_CMV & pSB1C3_betaglobin_mVenus</b></p>====
+
Investigator: Patrick
-
Investigator: <b>Chris L., Achim</B>
+
-
<br />
+
-
<p style="font-size:13px; color:#003399;"><b>Comments</b>: In order to proceed with the BioBrick assembly the plasmids pSB1C3_leftITR_CMV was cut with SpeI and PstI, pSB1C3_betaglobin_mVenus was cut with XbaI and PstI. The insert fragment containing betaglobin_mVenus was ligated to the vector fragment containing the leftITR_CMV promotor.</p>
+
-
<br />
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Harvesting cultures from the ViralBricks</b></p>====
-
<br />
+
Investigator: Volker
 +
 
 +
All approximately 150 cultures were pelletted (twice 2 ml) and the supernatant thrown away. The pellets were freezed and the rest of the culture stored in the 4°C room. These pellets will be preped, test digested and sequenced subsequently until right clones are identified.
 +
 
 +
===105. labday 30.08.2010===
 +
 
 +
====<p style="font-size:15px; background-color:#66bbff;"><b>BioBrick assembly of pSB1C3_leftITR_pTERT & pSB1C3_ß-globin_YFP_hGH_rITR</b></p>====
 +
 
 +
Investigator: Achim <br />
 +
 
 +
comment: final assembly of a vector containing all aav elements & the tert promoter <br />
<b>Digestion:</b> <br />
<b>Digestion:</b> <br />
{| border="1"
{| border="1"
-
| '''components'''   || align="right" |'''pSB1C3_leftITR_CMV''' || align="right" |'''pSB1C3_betaglobin_mVenus'''
+
| components:   || align="right" | pSB1C3_ß-globin_YFP_hGH_rITR(Vector): P294 || align="right" |pSB1C3_leftITR_pTERT (Insert): P256
|-
|-
-
| DNA  ||  align="right" |5,2 ||  align="right" |8,4  
+
| DNA  ||  align="right" |2,4 ||  align="right" |8,4  
|-
|-
| BSA (10x) ||  align="right" |2 ||  align="right" | 2  
| BSA (10x) ||  align="right" |2 ||  align="right" | 2  
Line 3,333: Line 1,594:
| Buffer 4 (10x)||  align="right" |2 ||  align="right" |2  
| Buffer 4 (10x)||  align="right" |2 ||  align="right" |2  
|-
|-
-
|Enzyme1 ||  align="right" |1 (SpeI) ||  align="right" |1 (Xba1)
+
|Enzyme1 ||  align="right" |1 (EcoRI) ||  align="right" |1 (EcoRI)
|-
|-
-
|Enzyme2 ||  align="right" |1 (PstI)||  align="right" |1 (PstI)  
+
|Enzyme2 ||  align="right" |1 (XbaI)||  align="right" |1 (SpeI)  
|-
|-
-
|H2O||  align="right" |8,8 ||  align="right" |5,6   
+
|H2O||  align="right" |11,6 ||  align="right" |5,6   
|-
|-
|'''Total volume'''||  align="right" | 20||  align="right" |20  
|'''Total volume'''||  align="right" | 20||  align="right" |20  
|}
|}
<br />
<br />
 +
Preparation of gel:<br />
Preparation of gel:<br />
-
0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 115 Volt, running time: 45 minutes
+
0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 110 Volt, running time: 45 minutes
<br />
<br />
 +
 +
 +
[[Image:Freiburg10 30082010achim2.JPG|200px]] <br />
 +
 +
Expected sizes of constructs:
Expected sizes of constructs:
-
*pSB1C3_leftITR_CMV:  2800 bp
+
*pSB1C3_ß-globin_YFP_hGH_rITR:  ~4000 bp
-
*pSB1C3_betaglobin_mVenus: 1200 bp, 2000 bp
+
*pSB1C3_leftITR_pTERT: 2896 bp, ~640 bp
The corresponding bands were cut out and Gel-Extraction was performed according to protocol.
The corresponding bands were cut out and Gel-Extraction was performed according to protocol.
Line 3,355: Line 1,622:
concentrations measured via NanoDrop:
concentrations measured via NanoDrop:
-
*pSB1C3_leftITR_CMV: 55,5 ng/µl
+
*pSB1C3_ß-globin_YFP_hGH_rITR: 34.73 ng/µl
-
*pSB1C3_betaglobin_mVenus:: 10,4 ng/µl
+
*pSB1C3_leftITR_pTERT: 9.48 ng/µl
-
<br />
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Continuation of Cloning of Rep40/52</b></p>====
+
<b>Ligation:</b> <br />
-
'''Investigator: Anna
+
T4 ligation was performed according to protocol. <br />
 +
Volumes used:
 +
*vector: 2,9 µl
 +
*insert: 5,1 µl
-
*Digestion of PCR products and vector:
 
-
<br />
+
<b>Transformation:</b> <br />
-
{| border="1"
+
-
| '''components'''  || align="right" |'''PCR product /µl''' || align="right" |'''vector /µl'''
+
-
|-
+
-
| DNA  ||  align="right" |19 ||  align="right" |10
+
-
|-
+
-
| BSA (10x) ||  align="right" |3 ||  align="right" | 3
+
-
|-
+
-
| Buffer 4 (10x)||  align="right" |3 ||  align="right" |3
+
-
|-
+
-
|Enzyme '''XbaI ||  align="right" |1 ||  align="right" |1
+
-
|-
+
-
|Enzyme '''SpeI ||  align="right" |1 ||  align="right" |1
+
-
|-
+
-
|H2O||  align="right" |3 ||  align="right" |12
+
-
|-
+
-
|'''Total volume (e.g. 15,20,25,30 µl)'''||  align="right" | 30||  align="right" |30
+
-
|}
+
-
<br />
+
-
<p style="font-size:13px; color:#68bbff;">'''''Comments''''': This time digestion was done with XbaI and SpeI (means it has to be checked if the inserts are cloned into the vector in the right orientation). </p> <br>
+
-
*Purification of Rep40 and Rep52:
+
bacterial strain used: XL1bB<br />
-
For the purification 95 µl of buffer PBI was used.
+
-
c(Rep40)= 12,5 ng/µl <br />
+
antibiotic used for plate: chlorampenicol<br />
-
c(Rep52)= 51,8  ng/µl <br />
+
 +
Transformation was performed according to protocol.<br />
 +
Plate was prepared and put in 37°C room.
-
<br />
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Continuation: Digestion of pSB1C3_NLS (P324)</b></p>====
-
*Gelextraction of pSB1C3_RFC25_CFP:
+
Investigator: Patrick
-
0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 120 Volt, running time:55 <br />
+
-
5µl loading dye (6x) for the sample, Marker: GeneRuler ladder mix (Fermentas)
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
[[file:Digestion of pSB1C3_2.jpg]]
+
[[File:Freigem10_Pat_3008.JPG|500px]]
-
<br />
+
 +
The gelextraction was performed with QIAEX II Gel Extraction Kit which should also allow to extract fragments smaller than 70 bp (at least 40 bp). According to the QIAGEN Gel Extraction Kit the lenght of the DNA fragment should be about 70 bp.
 +
Unfortunately the DNA fragment was blurred so a quite large piece had to be cut out.
-
c(pSB1C3)= 10,59 ng/µl <br />
+
The extraction yielded 4,26 and 3,77 ng/µl (two measurements) but this sample also quite polluted. ... put into freezer
-
c(pSB1C3_2)= 4,25 ng/µl
+
-
<br />
+
====<p style="font-size:15px; background-color:#66bbff;"><b>CD biobrick production</b></p>====
-
*Ligation of PCR products and vector:
 
-
For the Ligation 1µl T4 buffer (2x) and 1µl T4 ligase were used.
 
-
<br />
 
-
{| border="1"
 
-
| ''' '''  || align="right" |'''Sample-no.'''|| align="right" |'''vector /µl''' || align="right" |'''insert /µl'''
 
-
|-
 
-
| pSB1C3 + Rep40 ||  align="right" |1 || align="right" |3,65 ||  align="right" |4,35
 
-
|-
 
-
| pSB1C3 + Rep52 ||  align="right" |2 ||  align="right" |5,87 ||  align="right" |2,13
 
-
|-
 
-
|'''Total volume (e.g. 15,20,25,30 µl)'''||  align="right" | 8||  align="right" | ||  align="right" |
 
-
|}
 
-
<br />
 
-
*Transformation:
+
<b>Investigator: Kira</b><br>
 +
NA samples were diluted 1:100
-
The transformation was done following the standard protocol using B21 cells.
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>PCR of bla_14FM  and ligation into pSB1C3_SDM_SspI and pSB1C3_SDM_PvuII for virobrick-production T</b></p>====
 
-
<br/>
 
-
Investigator: Anissa,Kerstin<br />
 
-
<br />
 
-
<br />
 
-
<p style="font-size:13px; color:#68bbff;">'''''Comments''''': Because it still is unclear if SDM of pSB1C3_SspI_PvuII has worked, we decided to use the one time mutagenisised vector pSB1C3_SDM_SspI, to clone bla into it and to make the second mutation of PvuII after bla is inserted.  . </p> <br>
 
-
<br>
 
-
*'''PCR:
 
-
(was performed following the standard protocol)
 
<br>
<br>
{| border="1"
{| border="1"
-
| '''Ingredients''' || align="right" |'''v(pTB106_bla14FM) - DMSO / µl''' || align="right" |'''v(pTB106_bla14FM) + DMSO / µl'''
+
| '''Ingredients''' || align="right" |CD sample
|-
|-
-
| 5X Phusion HF buffer ||  align="right" |10 ||  align="right" |10
+
| 5X Phusion HF buffer ||  align="right" |10 µl
|-
|-
-
| 10 mM dNTP mix||  align="right" |1 ||  align="right" |1
+
| 10 mM dNTP mix||  align="right" |1µl
|-
|-
-
| forward primer: O116(P extreme f)||  align="right" |2,5 ||  align="right" |2,5
+
| forward primer: O158 ||  align="right" |2,5µl
|-
|-
-
| reverse primer: O117 (P extreme r) ||  align="right" |2,5 ||  align="right" |2,5
+
| reverse primer: O159 ||  align="right" |2,5 µl
|-
|-
-
| DNA Template 1ng ||  align="right" |5,3 µl ||  align="right" |5,3 µl
+
| DNA Template||  align="right" |0,5 µl
|-
|-
-
| DMSO (1%)||  align="right" | 0,5 ||  align="right" | 0,5
+
| DMSO ||  align="right" |0 µl
|-
|-
-
| Phusion Polymerase||  align="right" |0,5 ||  align="right" |0,5
+
| Phusion Polymerase||  align="right" |0,5 µl
-
|-
+
-
|H<sub>2</sub>O||  align="right" |28,2 ||  align="right" |27,7
+
|-
|-
-
|Total volume||  align="right" |50 ||  align="right" |50
+
|H<sub>2</sub>O||  align="right" |33µl
 +
|-
 +
|Total volume||  align="right" |50 µl
|}
|}
<br>
<br>
-
*''' PCR program:
+
PCR program:
-
 
+
{| border="1"
{| border="1"
-
|'''PCR Program'''|| align="right" |'''temperature/ °C''' |'''time'''
+
|Cycles||Temperature||Time
|-
|-
-
|1||  align="right" |98  || align="right" | 1'                             
+
|||98°C||1
|-
|-
-
|2|| align="right" |98 ||  align="right" | 15''
+
|8x||98°C||15"
|-
|-
-
|8x|| align="right" |61  ||  align="right" | 25''
+
|||60°C||25"
|-
|-
-
|3 || align="right" |72 ||  align="right" | 15''
+
|||72°C||60"
|-
|-
-
|4 || align="right" |98 ||  align="right" | 15''
+
|17x||98°C||15"
|-
|-
-
|5|| align="right" |72 ||  align="right" | 15''
+
|||65°C||25"
|-
|-
-
|1x|| align="right" |72 ||  align="right" | 5'
+
|||72°C||60"
|-
|-
-
|Hold|| align="right" |4
+
|1x||72°C||5'
 +
|-
 +
|Hold 4°C
|}
|}
<br>
<br>
-
[[File:Freiburg10 pcr bla14FM.JPG|300px]]
+
Digestion of plasmid backbone:
-
 
+
-
 
+
-
 
+
-
*'''Digestion PCR product:
+
-
 
+
-
 
+
-
 
+
-
* DNA-Concentration: 4,2 ng/µl
+
 +
c (pSB1C3) = 151, 1 ng/ µl
{| border="1"
{| border="1"
-
|'''components'''|| align="right" |'''volume / µl'''
+
| align="left" | '''Components''' ||align="left"| <b>vector</b> Volume/µL
|-
|-
-
| DNA || align="right" | 28,5                               
+
| align="left" | DNA 1 µg  ||align="left"| 6,0 µl
|-
|-
-
| BSA 100x || align="right" | 0,4
+
| align="left" | BSA (100x) ||align="left"| 0,2 µl
|-
|-
-
|Buffer: 4 10x || align="right" | 4 
+
| align="left" | Buffer no. 4 (10x) ||align="left"| 2,0 µl
|-
|-
-
| Enzyme 1 (XbaI) || align="right" | 1
+
| align="left" | Enzyme 1 XbaI ||align="left"| 0,5 µl
|-
|-
-
| Enzyme 2 (PstI) || align="right" | 1
+
| align="left" | Enzyme 2 AgeI HF ||align="left"| 0,5 µl
|-
|-
-
| H<sub>2</sub>O || align="right" | 5,1
+
| align="left" | H<sub>2</sub>O ||align="left"| 10,8 µl
-
|-
+
-
| Total volume ||  align="right" | 40
+
|-
|-
 +
| align="left" | '''Total volume''' ||align="left"| <b>20</b> 
|}
|}
 +
<br />
-
* ''' Purification of PCR product: (QlAquick Gel Extraction Kit)
+
incubation @ 37 C for approx. 2 h
-
+
-
preparation of purification was made according to the standardprotocol
 
-
 
+
Digestion of PCR product:
-
 
+
-
<p style="font-size:13px; color:#FF0033;">'''''Comments''''': The wrong enzymes were used. Experiment has to be done tomorrow.</p> <br>
+
-
 
+
-
====<p style="font-size:15px; background-color:#66bbFF;"><b>Test digestion pSB1C3_CFP_SDM SspI and PvuII </b></p>====
+
-
'''Investigator: Stefan'''<br>
+
-
*buffer used: 2; Restriction-enzymes used: Enzyme 1: ClaI ; Enzyme 2: BpmI ; Enzyme 3: SspI ; Enzyme 4: PvuII
+
-
<ul><li>Plasmids:</li>
+
-
<ul><li>pSB1C3_SDM_SspI/PvuII '''P185''' (first tree lanes, cut with different enzymes)</li>
+
-
<li>pSB1C3_RFC25_longlinker '''P105'''</li>
+
-
<li>pSB1C3_CFP '''P51.1'''</li>
+
-
</ul>
+
-
</ul>
+
-
 
+
-
<br />
+
{| border="1"
{| border="1"
-
| align="left" | '''Components''' ||align="left"| '''P185/µL''' ||align="left"| '''P185/µL''' ||align="left"| '''P185/µL''' ||align="left"| '''P105/µL''' ||align="left"| '''P51.1/µL'''
+
| align="left" | '''Components''' ||align="left"| <b>PCR product</b> Volume/µL  
|-
|-
-
| align="left" | DNA ||align="left"| 18||align="left"| 18||align="left"| 18||align="left"| 4,8||align="left"|6,8
+
| align="left" | DNA   ||align="left"| 30,0 µl
|-
|-
-
| align="left" | BSA (10x) ||align="left"| 2,5||align="left"| 2,5||align="left"| 2,5||align="left"| 2,5||align="left"| 2,5
+
| align="left" | BSA (100x) ||align="left"| 0,4 µl
|-
|-
-
| align="left" | Buffer 2 (10x) ||align="left"| -||align="left"| -||align="left"| -||align="left"| 2,5||align="left"| 2,5
+
| align="left" | Buffer no. 4 (10x) ||align="left"| 4,0 µl
|-
|-
-
| align="left" | Buffer 4 (10x)  ||align="left"| 2,5||align="left"| 2,5||align="left"| 2,5||align="left"| -||align="left"| -
+
| align="left" | Enzyme 1 XbaI ||align="left"| 1,5 µl
|-
|-
-
| align="left" | Enzyme 1 ClaI (152)  ||align="left"| 0,5||align="left"| 0,5||align="left"| 0,5||align="left"| -||align="left"| -
+
| align="left" | Enzyme 2 AgeI HF ||align="left"| 1,0 µl
|-
|-
-
| align="left" | Enzyme 2 BpmI (144)  ||align="left"| 0,5||align="left"| 0,5||align="left"| 0,5||align="left"| -||align="left"| -
+
| align="left" | H<sub>2</sub>O ||align="left"| 3,1 µl
|-
|-
-
| align="left" | Enzyme 3 SspI (68)  ||align="left"| -||align="left"| 0,5||align="left"| -||align="left"| 0,5||align="left"| 0,5
+
| align="left" | '''Total volume''' ||align="left"| <b>40</b> 
-
|-
+
-
| align="left" | Enzyme 4 PvuII (50) ||align="left"| -||align="left"| -||align="left"| 1||align="left"| 0,5||align="left"| 0,5
+
-
|-
+
-
| align="left" | H<sub>2</sub>O ||align="left"| 1||align="left"| -||align="left"| 0,5||align="left"| 14,2||align="left"| 12,2
+
-
|-
+
-
| align="left" | '''Total volume''' ||align="left"| 25||align="left"| 25||align="left"| 25||align="left"| 25||align="left"| 25
+
|}
|}
<br />
<br />
 +
incubation @ 37 C for approx. 2 h
-
*Incubation: 75 minutes<br>
+
1% agarose gel <br />
-
'''Agarosegel'''
+
-
<br />
+
-
0.5 g Agarose, 50 ml TAE, 3 µL GELRED, at 115 Volt, running time: 60 minutes
+
-
<br />
+
-
<br />
+
 +
Ligation <br />
 +
T4 ligase was used <br />
 +
1 ul T4 Buffer <br />
 +
1 ul T4 Ligase <br />
 +
9 ul (6,9 ul vector+ 1,1 ul insert) DNA-mix <br />
-
<br />
+
incubation over-night @ 18C <br />
-
[[File:Freiburg10 SspI PvuII ClaI BpmI later.png|500px|left|thumb|]]
+
 
 +
====<p style="font-size:15px; background-color:#66bbff;"><b>Repetition of test digestion of pSB1C3_Rep40_RC_Insert and pSB1C3_Rep68_RC_Insert.</b></p>====
 +
<b>Investigator: Chris L.</b><br>
 +
<ul>
 +
<li>P316 = pSB1C3_Rep40_ins clone 1<br>c=302,22 ng/µl</li>
 +
<li>P317 = pSB1C3_Rep40_ins clone 2<br>c=274,37 ng/µl</li>
 +
<li>P318 = pSB1C3_Rep68_ins clone 1<br>c=491,69 ng/µl</li>
 +
<li>P319 = pSB1C3_Rep68_ins clone 2<br>c=498,71 ng/µl</li>
 +
</ul>
<br>
<br>
 +
<b>Test digestion:</b>
<br>
<br>
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
<br>
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
<br>
 
-
comments: Test digestion of pSB1C3_SDM_SspI/PvuII with ClaI, BpmI and SspI looks the same as with ClaI and BpmI. Therefore we can assume that there is no additional restriction site within the vector. But still the bands above 3000 bp cannotz be explained. A new test digestion will be performed tomorrow to check the size of the vector.<br />
 
-
The approach cut with ClaI, BpmI and PvuII looks strange. Propably, PvuII does not work correctly. New PvuII-HF was ordered and delivered so this can be verified tomorrow.<br />
 
-
pSB1C3_RFC25_longlinker digested with SspI and PvuII seems to be not completely digested This could correspond to non-working PvuII.
 
-
pSB1C3_CFP looks like expected.
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>pSB1C3_SDM_Ssp1 (P125) sent for sequenzing</b></p>====
 
-
<br/>
 
-
Investigator: Stefan<br />
 
-
 
-
comment: To verify the correct deletion of the SspI restriction site it was sent for sequenzing.
 
-
 
-
Primer used: GATC_std_pTeSp-2
 
-
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Picking clones of pSB1C3_SDM_SspI/PvuII (P185)</b></p>====
 
-
<br/>
 
-
Investigator: Adrian<br />
 
-
 
-
comment: pSB1C3_SDM_SspI/PvuII (P185) is empty. For plasmid production DYT + Cm was inoculated from the glycerol stock (B159).<br>
 
-
 
-
=== 87. labday 12.08.2010===
 
-
====<p style="font-size:15px; background-color:#FF0033;"><b>XL1blue are ready to use</b></p>====
 
-
'''Investigator: Jessica'''
 
-
<br>there are 60µl aliquots to use for one trafo
 
-
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-Prep of pSB1C3_SDM_SspI/PvuII and pMA_RC_insert</b></p>====
 
-
<br/>
 
-
Investigator: Stefan<br />
 
-
 
-
Gycerol stock was prepared for pMA_RC_insert and labeled B167. No glyerol stock for pSB1C3_SDM_SspI/PvuII because cells from earlier stock werer used for Mini-Prep.
 
-
 
-
Miniprep was performed according to protocol.<br />
 
-
 
-
Samples were labeled:<br />
 
-
*pSB1C3_SDM_SspI/PvuII = P185.1
 
-
*pMA_RC_insert = P190
 
-
Concentrations:
 
-
*pSB1C3_SDM_SspI/PvuII (P185.1): 257,2 ng/µl
 
-
*pMA_RC_insert (P190): 339,5 ng/µl
 
-
<br />
 
-
No test digestion was performed.
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Biobrick Assembly: pSB1C3_lITR_CMV & pSB1C3_betaglobin_mVenus</b></p>====
 
-
Investigator: <b>Chris L., Achim</B>
 
-
<br />
 
-
<p style="font-size:13px; color:#003399;"><b>Comments</b>: In order to proceed with the BioBrick assembly the plasmids pSB1C3_leftITR_CMV was cut with SpeI and PstI, pSB1C3_betaglobin_mVenus was cut with XbaI and PstI. The insert fragment containing betaglobin_mVenus was ligated to the vector fragment containing the leftITR_CMV promotor.</p>
 
-
 
-
<br />
 
-
 
-
<b>Ligation:</b> <br />
 
-
For ligation, T4 ligase was used.
 
-
<br />
 
-
* 1µl T4-Ligase buffer (10x)
 
-
* 8µl (Vector + Insert) mix
 
-
* 1µl T4-Ligase
 
-
Ligation was carried out as following:
 
-
*pSB1C3_leftITR_CMV: 6,98 µl
 
-
*pSB1C3_betaglobin_mVenus: 1,02 µl
 
-
 
-
<br /> Incubation for 40 minutes.
 
-
 
-
*Transformation:
 
-
 
-
The transformation was done following the standard protocol using XL1B cells.
 
-
 
-
<br />
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Repetition of cloning of bla14FM_viralbrick_MCS into pSB1C3_SDM_SspI</b></p>====
 
-
<br/>
 
-
Investigator: Anissa<br />
 
-
<p style="font-size:13px; color:#003399;"><b>Comments</b>:Yesterday the pcr-product of bla14FM was cut with the wrong enzymes, so no overhangs could result. Today it will be repeated with the rihgt ones (SpeI and NgomIV) .</p>
 
-
 
-
 
-
 
-
*'''Digestion of the PCR product bla14FM for 2 h :
 
-
 
-
 
-
 
-
* DNA-Concentration: 10,2 ng/µl
 
-
 
-
 
{| border="1"
{| border="1"
-
|'''components'''|| align="right" |'''volume / µl'''  
+
| components ||volume of '''P316'''/µl ||volume of '''P317'''/µl ||volume of '''P318'''/µl ||volume of '''P319'''/µl
|-
|-
-
| DNA || align="right" | 18                               
+
| DNA || 1 || 1 || 1 || 1
|-
|-
-
| BSA 100x || align="right" | 3
+
| BSA (10x) ||1 ||1 || 1 || 1
|-
|-
-
|Buffer: 4 10x || align="right" | 3 
+
| Buffer 4 (10x) ||1 ||1 || 1 || 1
|-
|-
-
| Enzyme 1 (NgomIV) || align="right" | 1
+
|Enzyme XcmI ||0,5 ||0,5 ||0,5 || 0,5
|-
|-
-
| Enzyme 2 (SpeI) || align="right" | 1
+
|H2O || 6,5 || 6,5 || 6,5 || 6,5
-
|-
+
-
| H<sub>2</sub>O || align="right" | 4
+
-
|-
+
-
| Total volume ||  align="right" | 30
+
|-
|-
 +
|'''Total volume /µl'''||10 ||10 ||10 ||10
|}
|}
 +
<br />
 +
Incubation time: 1 h, Incubation temperature: 37°
 +
>br />
 +
Preparation of gel:<br />
 +
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 120 Volt, running time: 45 minutes
 +
<br />
 +
[[File:Freiburg10 pSB1C3 Rep40 ins and pSB1C3 Rep68 ins.png|600px]]
 +
<br />
 +
<p style="font-size:13px; color:#003399;"><b>Comment</b>: Test digestion looks good. Clones 2 of each plasmid (P317 and P319) were sent in for sequencing with VR2 Primer. </p>
-
* ''' Purification of PCR product: (QlAquick Gel Extraction Kit)
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Midi-Prep of pAAV_RC_insertparts clone1</b></p>====
-
+
-
preparation of purification was made according to the standardprotocol
+
'''Investigators: Chris W. <br>
-
<br/>
+
<p style="font-size:13px; color:#003399;"><b>Comment</b>: Midi-Preps of B208</p>
-
 
+
The Midi-Preps were performed according to the standard protocol yielding the following concentrations:
-
*'''Digestion  of vector backbone pSB1C3_SDM_SspI for 2 h:
+
-
<br/>
+
-
 
+
-
*1 µg vector has been used
+
{| border="1"
{| border="1"
-
|'''components'''|| align="right" |'''volume / µl'''
+
| plasmid-no. || align="right" |P325|| align="right" |p326
|-
|-
-
| DNA ||  align="right" | 3,12                             
+
| concentration (ng/µl)|| align="right" |2481,04 || align="right" |2050,66
-
|-
+
-
| BSA 100x ||  align="right" | 1,5
+
-
|-
+
-
|Buffer: 4 10x ||  align="right" | 1,5
+
-
|-
+
-
| Enzyme 1 (NgomIV) || align="right" | 1
+
-
|-
+
-
| Enzyme 2 (SpeI) ||  align="right" | 1
+
-
|-
+
-
| H<sub>2</sub>O ||  align="right" | 6,88
+
-
|-
+
-
| Total volume ||  align="right" | 15
+
|-
|-
|}
|}
-
<br/>
+
<br>
-
*the cut vector was loaded on a preperative 1% agarose gel and the gel was running for 45 minutes
+
====<p style="font-size:15px; background-color:#66bbff;"><b>FACS analysis, Seeding HT1080 and AAV293</b></p>====
 +
'''Investigators: Kerstin, Adrian
 +
*FACS analysis of Transduction from 28.08.2010
 +
*Seeding HT1080 cells for Transduction (31.08.2010): 5x 6-well plates, 270.000 cells per well
 +
*Seeding AAV293 cells for Transfection (01.09.2010): 10 plates, 300.000 cells per well
-
[[File:Freiburg10 pSB1C3 SDM SspI cut.png|400px]]
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-Preps and test digestion of Loop insertion BioBricks</b></p>====
-
<br/>
+
Investigator: Anna
-
*gel was cut and gelextraction was performed according the qiagen standard protocol.
+
-
<br/>
+
-
*'''Ligation of pSB1C3_SDM_SspI and bla14FM:
+
<p style="font-size:13px; color:#003399;"><b>Comment</b>: Two clones of each ViralBrick were prepared and test digested. </p>
-
<br/>
+
-
{| border="1"
+
-
|'''components'''|| align="right" |'''volume / µl'''
+
-
|-
+
-
|t4 buffer (10X)  ||  align="right" | 1                             
+
-
|-
+
-
| t4 Ligase ||  align="right" | 1
+
-
|-
+
-
|vector DNA ||  align="right" | 2,72
+
-
|-
+
-
| Insert ||  align="right" | 5,28
+
-
|-
+
-
|}
+
-
<br/>
+
-
*'''Transformation: pSB1C3_SDM_SspI_bla14FM was transformed into BL21 following the stanard protocol
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Quickchange of the EaglII restriction site in the PMA_RepCap vector</b></p>====
+
<b>Mini-Preps:</b>
-
Investigator: <b>Chris L., Achim</B>
+
-
<br />
+
-
<p style="font-size:13px; color:#003399;"><b>Comments</b>: We performed a QuikChange mutagenesis on the synthesized PMA_RepCap vector (P190). The EaglII restriction enzyme also cuts NotI (IGEM standard) therefore EaglII has to be changed into a different recognition site. Primers were designed to change it into PvuII. The new QuikChange Lighting Site-Directed Mutagenesis Kit from Stratagene was used, therefore the mutagenesis was carried out as described in the QuikChange Manual. The PCR product was transformed into XL10-Gold cells, we used the IGEM standard protocol, except for the addition of ß-ME to the XL10-Gold cells.  : </p>
+
-
PCR-reaction:
 
{| border="1"
{| border="1"
-
| '''Volume / µl''' || align="right" |'''ingredients'''|| align="right" |'''recommended /µl'''
+
| || align="right" |'''p331''' || align="right" |'''p335'''|| align="right" |'''p339'''|| align="right" |'''p342'''|| align="right" |'''p345'''
 +
 
|-
|-
-
| 2,5 ||  align="right" |10X reaction buffer ||  align="right" | 2,5
+
|Construct||  align="right" |pSB1C3_587_KO_BAP_clone3.1 ||  align="right" | pSB1C3_587_RGD_clone5.1 ||  align="right" | pSB1C3_453_His_clone7.1||  align="right" | pSB1C3_587_His_clone8.3  ||align="right" |pSB1C3_587_KO_empty_clone10.1                           
|-
|-
-
| 2,9 ||  align="right" |P190 template (~10 ng)||  align="right" | 2,9 of 1:100 dilution
+
|DNA-Concentration / µl||  align="right" |65,08 ||  align="right" | 94,29||  align="right" | 73,49||  align="right" |69,05||  align="right" | 66,73
|-
|-
-
| 0,51||  align="right" |forward primer: O122||  align="right" | 62,5 ng
 
-
|-
 
-
|0,51 ||  align="right" |reverse primer: O123 ||  align="right" | 62,5 ng
 
-
|-
 
-
|- ||  align="right" |DMSO ||  align="right" | -
 
-
|-
 
-
|0,5||  align="right" |dNTP||  align="right" | 250 µM each dNTP
 
-
|-
 
-
|17,6||  align="right" |H<sub>2</sub>O ||  align="right" |
 
-
|-
 
-
|0,5||  align="right" |QuikChange Lightning ennzyme(1.25) ||  align="right" |
 
-
|}
 
-
<br>
 
-
PCR program:
 
-
{| border="1"
 
-
|'''Rounds'''|| align="right" |'''temperature/ °C'''|| align="right" |'''Time'''
 
-
|-
 
-
|1 ||  align="right" |95 ||  align="right" | 2 minutes
 
-
|-
 
-
|18 ||  align="right" |95||  align="right" |20 seconds
 
-
|-
 
-
|18 ||  align="right" |60 ||  align="right" | 10 seconds
 
-
|-
 
-
|18||  align="right" |68||  align="right" | 2 minutes (4 kb * 30 sec)
 
-
|-
 
-
|1||  align="right" |68||  align="right" | 5 minutes
 
|}
|}
-
<br>
+
<b>Test digestion:</b>
-
The PCR product was digestet with Dpn I for 10 minutes at 37°C
+
-
Plasmid was transformed into XL10-Gold cells.
+
[[File:Freiburg10_LoopInsertions_Sal_Bam.jpg|600px]]
-
<br/>
+
 
 +
1% Agarose gel: 1 g Agarose, 100 ml TAE, 6 µl Gelred <br/>
 +
Marker: Gene ruler ladder DNA mix, 5 µl<br/>
-
====<p style="font-size:15px; background-color:#ff00ff;"><b>Sequencing results of Rep68, Rep78 and AAP BioBricks</b></p>====
+
Used enzymes: SalI, BamHI (in general, SDS has to be added because of the strong binding of enzymes to DNA!)
-
Investigator: <b>Hanna</B>
+
-
<br />
+
-
Comment: pSB1C3_Rep78 (P160), pSB1C3_Rep68 (P165) and pSB1C3_AAP (P170) were sent for sequencing twice. No sequencing made sense. Paradoxically the Rep78 sequencing result fit to the p5 promoter BioBrick... <br/>
+
-
By having a closer look to the BioBrick PCR we found out that the fragment sizes didn't fit to the expected sequences. In addition to that the test digestion also showed some discrepancies. <br/>
+
-
Because we weren't able to find out in which steps of the BioBrick production things went wrong, we decided to discard all referring glycerol stocks and plasmids and to perform the Rep68, Rep78 and AAP BioBrick PCR again!!!
+
<br/>
<br/>
<br/>
<br/>
-
====<p style="font-size:15px; background-color:#9acd32;"><b>Comment 13.08.2010 (Hanna):</b> We wondered how the whole Rep stuff could be sequenced reverse AND forward without any forward sequencing primer... By checking the "sequencing book" we found out that always the wrong primers were used: the GOI primers can be used in order to sequence the <b>G</b>ene <b>o</b>f <b>i</b>nterest = GOI (!) in pAAV_MCS and NOT the pSB1C3!!!!!!!!!</p>====
+
<p style="font-size:13px; color:#003399;"><b>Comment</b>: Test digestion had no significant results. In addition, one sample was forgotten, it was not clear which one (description makes no sense). Test digestion has to be repeated.</p>
 +
<br/>
<br/>
<br/>
-
====<p style="font-size:15px; background-color:#66bbFF;"><b>Order oligos for CD-biobrick</b></p>====
+
====<p style="font-size:15px; background-color:#ff00ff;"><b>Repetition of subcloning Cap-insert into pAAV_RC</b></p>====
-
'''Investigator: Jessica and Kira'''
+
Investigator: Stefan
-
<br>ordered on aug 12th<br>
+
-
[[File:Freiburg10 oligos for CD-biobrick.jpg]]
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>BioBrick assembly of pSB1C3_leftITR_CMV_mVenus</b></p>====
+
<p style="font-size:13px; color:#003399;"><b>Comments</b>: There are still problems with BspMI. It was assumed that longer digestion could improve the results, therefore digestion with BspMI will be performed overnight.<br />
-
 
+
<b>P.S: Welcome back Hanna!</b></p>
-
Investigator: Stefan <br />
+
-
 
+
-
comment: In order to assemble our complete constructs mVenus has to be included into the pSB1C3_leftITR_CMV vector. <br />
+
-
<b>Digestion:</b> <br />
+
-
{| border="1"
+
-
| components:  || align="right" |pSB1C3_leftITR_CMV P188(Vector) || align="right" |pGA14_mVenus P60 (Insert)
+
-
|-
+
-
| DNA  ||  align="right" |6 ||  align="right" |8
+
-
|-
+
-
| BSA (10x) ||  align="right" |2 ||  align="right" | 2
+
-
|-
+
-
| Buffer 4 (10x)||  align="right" |2 ||  align="right" |2
+
-
|-
+
-
|Enzyme1 ||  align="right" |1 (SpeI) ||  align="right" |1 (Xba1)
+
-
|-
+
-
|Enzyme2 ||  align="right" |1 (PstI)||  align="right" |1 (PstI)
+
-
|-
+
-
|H2O||  align="right" |8 ||  align="right" |6 
+
-
|-
+
-
|'''Total volume'''||  align="right" | 20||  align="right" |20
+
-
|}
+
<br />
<br />
-
 
-
 
-
Preparation of gel:<br />
 
-
0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 110 Volt, running time: 45 minutes
 
<br />
<br />
-
 
+
'''Digestion of vector and insert:'''
-
 
+
*Insert: pMA_RepCap Vector_SDM_InsPvuII clone 1 (P211)
-
[[File:Freiburg10 cloning of pSB1C3 leftITR CMV mVenus.png|500px]] <br />
+
*Vector: pAAV_RC_ins-rep clone1 (P250)<br />
-
 
+
-
 
+
-
 
+
-
Expected sizes of constructs:
+
-
*pSB1C3_leftITR_CMV: 2869 bp
+
-
*pGA14_mVenus: 2896 bp, 756 bp
+
-
 
+
-
The corresponding bands were cut out and Gel-Extraction was performed according to protocol.
+
<br />
<br />
-
 
-
concentrations measured via NanoDrop:
 
-
*pSB1C3_leftITR_CMV: 39,2 ng/µl
 
-
*pGA14_mVenus: 8,2 ng/µl
 
-
 
-
 
-
<b>Ligation:</b> <br />
 
-
 
-
T4 ligation was performed according to protocol. <br />
 
-
 
-
Volumes used:
 
-
*vector: 1,67 ng/µl
 
-
*insert: 6,33 ng/µl
 
-
 
-
 
-
<b>Transformation:</b> <br />
 
-
 
-
bacterial strain used: XL1bB<br />
 
-
 
-
antibiotic used for plate: chlorampenicol<br />
 
-
 
-
Transformation was performed according to protocol.<br />
 
-
Plate was prepared and put in 37°C room.
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Opened a BioBrick box</b></p>====
 
-
<b>Investigator: Bea</b>
 
<br />
<br />
-
In order to "save" the plasmids/BioBricks which has been prepared and confirmed for submitting it to the parts registry a new box have been prepared. The box should contain 30 µL of the desired construct cloned into the pSB1C3 vector and the BBa number given from the headquarter. <br />
+
A two-step digestion was performed: <br />
-
 
+
-
[[File:Freiburg10 BioBrick highlighting in Nomenclature list.JPG|400px|thumb]]
+
-
The first BioBricks which are in this box are:
+
-
<ul>
+
-
<li>pSB1C3_hGH (P136): Bba_K404002</li>
+
-
<li>pSB1C3_betaglobin (P133: Bba_K404001 </li>
+
-
<li>pSB1C3_CMV (P145): Bba_K404003</li>
+
-
<li>pSB1C3_leftITR (P175): Bba_K404006</li>
+
-
<li>pSB1C3_rightITR (P172): Bba_K404005</li>
+
-
<li>pSB1C3_mGMK (P156): Bba_K404004</li>
+
-
</ul>
+
-
In our nomenclature plasmid excel list the constructs which we aliquoted should be highlighted in blue and commented with the given Bba number. Additionally it should be taken in consideration that enough DNA should be in the "working" plasmid box. Therefore, if necessary, inoculate DYT medium for Mini-Preps.<br />
+
<br />
<br />
-
In this case ALL the glycerol stocks were used  for incoluating 10 mL DYT medium containing 10 µL chloramphenicol respectively.
+
'''1st step'''
-
<ul>
+
-
<p style="color:#66bbff;">To do: Mini-Preps of the over night cultures.</p>
+
-
</ul>
+
-
<br/>
+
-
 
+
-
=== 88. labday 13.08.2010===
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Continuation:pSB1C3_SDM_SspI_bla14FM</b></p>====
+
-
Investigator: Patrick <br>
+
-
Intention: receive enough plasmid for a sequencing and maybe futher working steps. <br>
+
-
Three clones were picked in the morning to inoculate DYT and perform a mini-prep in the evening and to send them for sequencing. The three clones were picked and prepped according to the standard protocol yielding the following DNA concentrations:
+
-
*pSB1C3_SDM_SspI_bla14FM clone 1: 65,15 ng/µl , Sequencing labeled: PS3.1
+
-
*pSB1C3_SDM_SspI_bla14FM clone 2: 22,3 ng/µl , Sequencing labeled: PS3.2
+
-
*pSB1C3_SDM_SspI_bla14FM clone 3: 62,3 ng/µl , Sequencing labeled: PS3.3
+
-
<br>
+
-
Used primer: seq_pSB1C3_VR2_rev (O51) <br>
+
-
Results: see http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal#Sequence_analysis_of_pSB1C3_SDM_SspI_BLA
+
-
 
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-preps of pSB1C3_hgh,pSB1C3_beta globin,pSB1C3_left ITR,pSB1C3_right ITR,pSB1C3_mGMK,pSB1C3_CMV</b></p>====
+
-
<br/>
+
-
<p style="font-size:13px; color:#003399;"><b>Comments</b>:new mini-preps of the glycerol-stocks ofpSB1C3_hgh(=B103),pSB1C3_beta globin(=B100),pSB1C3_left ITR (=B143),pSB1C3_right ITR (=B140),pSB1C3_mGMK (=B125),pSB1C3_CMV  : </p>
+
-
<br/>
+
{| border="1"
{| border="1"
-
|'''components'''|| align="right" |'''concentration in ng/µl'''  
+
| || align="right" |'''P211 / µl''' ||'''P250 / µl'''
|-
|-
-
| P191 (pSB1C3_right ITR)||  align="right" | 100,74                               
+
|DNA||  align="right" |8,5  ||  align="right" | 2                             
|-
|-
-
|P192 (pSB1C3_beta-globin) ||  align="right" |240,08
+
|buffer 3||  align="right" |4,6 ||  align="right" | 5
|-
|-
-
|P193 (pSB1C3_CMV) ||  align="right" | 333,89  
+
| Enzyme BsiWI||  align="right" |1 ||  align="right" | 1
|-
|-
-
|P194 (pSB1C3_hgh) ||  align="right" |328,42
+
|H<sub>2</sub>O||  align="right" |32,4 ||  align="right" | 38
|-
|-
-
| P195 (pSB1C3_mGMK) ||  align="right" |258,63
+
|total volume||  align="right" |46 ||  align="right" |46
 +
|}
 +
<br />
 +
'''2nd step'''
 +
{| border="1"
 +
| || align="right" |'''P211 / µl''' ||'''P250 / µl'''
|-
|-
-
| P196 (pSB1C3_left ITR) ||  align="right" |173,98
+
|Mix obtained from step 1 ||  align="right" |46  ||  align="right" | 46                             
|-
|-
 +
| Enzyme BspMI||  align="right" |4  ||  align="right" | 4
 +
|-
 +
|total volume||  align="right" |50 ||  align="right" |50
|}
|}
-
 
+
<br />
-
====<p style="font-size:15px; background-color:#FF00ff;"><b>Design and ordering of oligos for VP fusion targeting approaches</b></p>====
+
<br />
-
'''Investigator: Hanna'''
+
Digestion will be performed overnight and continued tomorrow.
-
<br/>
+
-
<p> <b>Comment:</b> In order to target the EGFR receptor of our A431 cells different approaches have been figured out: Besides loop insertions, N-terminal fusion to the VP2 protein will be performed. In addition to that a VP1 insertion approach (after Grieger et al., 2007) was planned. <br/>
+
-
Fusion and insertion molecules will be e.g. His-tag, Affibody, Darpin, GFP. These approaches can be combinated with the loop insertions. <br/>
+
-
For this purpose the following oligos were ordered today: <br/></p>
+
-
<br/>
+
-
[[File:Freiburg10 N-terminalFusion1.jpg|450px|thumb|left|]]
+
-
[[File:Freiburg10 N-terminalFusion2.jpg|450px|thumb|right|]]
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
-
<br/>
+
<br/>
<br/>
<br/>
<br/>
 +
===106. labday 31.08.2010===
 +
====<p style="font-size:15px; background-color:#ff00ff;"><b>Cloning ZEGFR:1907 and 6xHis_middlelinker into pCerulean</b></p>====
 +
Investigator: Hanna
<br/>
<br/>
 +
<p><font color="#ff00ff"><b>Comment:</b> In order to fuse the His-Tag to VP2, the His-Tag-Middlelinker construct will be cloned into the pCerulean plasmid (contains CMV promoter and SV40 terminator) today. In the next step, VP2 will be cloned behind the His-Tag-Middlelinker.
<br/>
<br/>
 +
In addition to that the Affibody (ZEGFR:1907) will be also cloned into pCerulean (without linker). Also here, the next step will be fusing VP2 behind the this targeting molecule.</font></p>
<br/>
<br/>
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-Prep and Test digestion of Rep40 and Rep52 of the cloning experiment ("Cloning of Rep40/52" 11.08.2010 Investigator: Anna) </b></p>====
+
<p style="font-size:15px; font-weight: bold; color: #ff00ff;"><u>Practical Cloning:</u></p>
-
Investigator: <b>Chris L., Achim</B>
+
*plasmid:  
 +
**Vector: name: pCerulean; number: P273
 +
**Insert: name: pSB1C3_6xHis_middlelinker (P310) <b>+</b> pSB1C3_Zegfr:1907 (because P267 was empty, P285 was used! See lab day 23.08.!)
 +
*new vector name: pCerulean_6xHis_middlelinker <b>+</b> pCerulean_Zegfr:1907
 +
*buffer used: 4; Restriction-enzymes used: XbaI and PstI
 +
<br />
 +
'''Comments:''' Because the P267 tube was empty (!), P285 was used which was prepared by Jessy - I don't know whether this is a trafo of the glycerol stock? Sequenced?
<br />
<br />
-
 
+
<br />
-
<p style="font-size:13px; color:#003399;"><b>Comments</b>: There are small and big colonies on the plate. Maybe problems with the agar plate. Picked colonies of pSB1C3_Rep40 sample 1 and pSB1C3_Rep52 sample 1 are small colonies. Picked colonies of pSB1C3_Rep40 sample 2 + 3 and pSB1C3_Rep52 sample 2 + 3 are standard size colonies.</p>
+
<p style="font-size:15px; font-weight: bold; color: #ff00ff;">Digestion</p>
-
<br/>
+
-
 
+
-
'''Nanodrop'''
+
-
* Rep 40 Sample 1: 156,26 ng/µl '''P197'''
+
-
* Rep 40 Sample 2: 200,05 ng/µl '''P198'''
+
-
* Rep 40 Sample 3: 144,06 ng/µl '''P199'''
+
-
* Rep 52 Sample 1: 173,36 ng/µl '''P200'''
+
-
* Rep 52 Sample 2: 160,75 ng/µl '''P201'''
+
-
* Rep 52 Sample 3: 295,77 ng/µl '''P202'''
+
-
 
+
-
<br>
+
-
<br>
+
-
'''Test digestion''' 800ng DNA
+
{| border="1"
{| border="1"
-
| components  || align="right" |P145 /µl || align="right" |P146 /µl || align="right" |P145 /µl || align="right" |P146 /µl|| align="right" |P145 /µl || align="right" |P146 /µl
+
| components  || align="right" |volume of pCerulean/µl || align="right" |volume of pSB1C3_6xHis_middlelinker/µl || align="right" |volume of pSB1C3_ZEGFR:1907
|-
|-
-
| DNA  ||  align="right" | 5,1||  align="right" |4||  align="right" |5,6||  align="right" |4,6||  align="right" |5||  align="right" |2,7
+
| DNA  ||  align="right" | 3.6 ||  align="right" | 16.2 ||  align="right" | 11.5
|-
|-
-
| BSA (10x) ||  align="right" | 2||  align="right" |2||  align="right" |2||  align="right" |2||  align="right" |2||  align="right" |2
+
| BSA (10x) ||  align="right" | 3 ||  align="right" | 2.5 ||  align="right" | 2
|-
|-
-
| Buffer 4 (10x)||  align="right" | 2||  align="right" |2||  align="right" |2||  align="right" |2||  align="right" |2||  align="right" |2
+
| Buffer 4 (10x)||  align="right" | 3 ||  align="right" | 2.5 ||  align="right" | 2
|-
|-
-
| Enzyme 1: XbaI ||  align="right" | 0,5||  align="right" |0,5||  align="right" |0,5||  align="right" |0,5||  align="right" |0,5||  align="right" |0,5
+
|Enzyme 1 XbaI ||  align="right" | 1.5 ||  align="right" | 1 ||  align="right" | 1
|-
|-
-
| Enzyme 2: SpeI ||  align="right" | 0,5||  align="right" |0,5||  align="right" |0,5||  align="right" |0,5||  align="right" |0,5||  align="right" |0,5
+
|Enzyme 2 PstI ||  align="right" | 1.5 ||  align="right" | 1 ||  align="right" | 1
|-
|-
-
|H<sub>2</sub>O||  align="right" | 9,9||  align="right" |11||  align="right" |9,4||  align="right" |10,4||  align="right" |10||  align="right" |12,3
+
|H<sub>2</sub>O||  align="right" | 17.4 ||  align="right" | 1.8 ||  align="right" | 2.5
|-
|-
-
|'''Total volume (e.g. 15,20,25,30 µl)'''||  align="right" | '''20'''||  align="right" | '''20'''||  align="right" | '''20'''||  align="right" | '''20'''||  align="right" | '''20'''||  align="right" | '''20'''
+
|'''Total volume'''||  align="right" | 30 ||  align="right" | 25 ||  align="right" | 20
|}
|}
-
<br>
+
*Incubation: 1.5 h
-
Incubation time: 45 minutes, Incubation temperature: 37°
+
<br /><br />
-
<br>
+
-
<br>
+
-
<br>
+
-
0,5 g Agarose, 50ml x TAE, 3µl GELRED, at 115 Volt, running time: 45 minutes
+
-
 
+
 +
<p style="font-size:15px; font-weight: bold; color: #ff00ff;">Agarose-Gel:</p>
 +
<br />
 +
0.8 g Agarose, 55 ml TAE (1.45 %), 3 µL GELRED, at 115 Volt, running time: 35 minutes
 +
<br />
 +
<br />
{| border="1" cellspacing="0" cellpadding="2" bordercolor="black"
{| border="1" cellspacing="0" cellpadding="2" bordercolor="black"
!Sample
!Sample
Line 4,047: Line 1,951:
!Loading dye (6x)/µl
!Loading dye (6x)/µl
!Expected size 1 (Geneious)
!Expected size 1 (Geneious)
-
!Expected size 2 (Geneious)
 
|--
|--
-
|Rep 40 1
+
|P285
|20 µl  
|20 µl  
-
| 4 µl
+
|4 µl
-
|____ bp
+
|210 bp
-
| 940 bp
+
|--
|--
-
|Rep 40 2
+
|P273
-
|20 µl  
+
|30 µl  
-
| 4 µl
+
|6 µl
-
|____ bp
+
|~ 3900 bp
-
| 940 bp
+
-
|--
+
-
|Rep 40 3
+
-
|20 µl
+
-
| 4 µl
+
-
|____ bp
+
-
| 940 bp
+
-
|--
+
-
|Rep 52 1
+
-
|20 µl
+
-
| 4 µl
+
-
|____ bp
+
-
|1198 bp
+
-
|--
+
-
|Rep 52 2
+
-
|20 µl
+
-
| 4 µl
+
-
|____ bp
+
-
|1198 bp
+
-
|--
+
-
|Rep 52 3
+
-
|20 µl
+
-
| 4 µl
+
-
|____ bp
+
-
|1198 bp
+
|--
|--
 +
|P310
 +
|25 µl
 +
|5 µl
 +
|86 bp
 +
|}
|}
{| align=right
{| align=right
|}
|}
-
 
+
<br />
-
 
+
*Marker: GeneRuler ladder mix
{| border="1"
{| border="1"
|
|
-
!Marker
+
!Marker /µL
-
!Sample Rep40 1 /20µl
+
!Sample P285 /µl
-
!Sample Rep40 2 /20µl
+
!Sample P273 /µl
-
!Sample Rep40 3 /20µl
+
!Sample P310 /µl
-
!Sample Rep52 1 /20µl
+
-
!Sample Rep52 2 /20µl
+
-
!Sample Rep52 3 /20µl
+
|-
|-
!Lane  
!Lane  
-
| 1
+
|5.5
-
| 2
+
|24
-
| 3
+
|25
-
| 4
+
|25
-
| 5
+
-
| 6
+
-
| 7
+
|-
|-
|}
|}
-
 
-
agarose gel: 1%, digestion: 45 minutes, 37°C
 
-
 
-
 
-
<p style="font-size:13px; color:#68bbff;">'''''Comments:'''''Results look bad. Rep52 clone 3 looks good, send for sequencing. </p><br>
 
-
'''</p>
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Repetition of Rep 40 and Rep 52 ligation and transformation with the vector pSB1C3_CFP</b></p>====
 
-
 
-
Investigator: <b>Chris L.</B>
 
<br />
<br />
-
 
+
[[File:Freiburg10_Digestion_6xHis_middlelinker_and_Zegfr.JPG.gif|700px]]
-
*Ligation of PCR products and vector:
+
<p style="font-size:15px; font-weight: bold; color: #ff00ff;">Gel extraction</p>
-
 
+
<br />
-
For the Ligation 1µl T4 buffer (10x) and 1µl T4 ligase were used.
+
Gel measurement:
<br />
<br />
{| border="1"
{| border="1"
-
| ''' '''   || align="right" |'''Sample-no.'''|| align="right" |'''vector /µl''' || align="right" |'''insert /µl'''  
+
| align="left" | '''Sample'''  
 +
| align="left" | '''Weight'''  
 +
| align="left" | '''Volume'''  
 +
| align="left" | '''Concentration'''  
|-
|-
-
| pSB1C3 + Rep40 || align="right" |1 || align="right" |4,92 || align="right" |3,08
+
| align="left" | P285
 +
| align="left" | 240 mg
 +
| align="left" | 20 µL
 +
| align="left" | 5.4 ng/µL
|-
|-
-
| pSB1C3 + Rep52 || align="right" |2 ||  align="right" |6,47 || align="right" |1,53
+
| align="left" | P273
 +
| align="left" | 130 mg
 +
| align="left" | 20 µL
 +
| align="left" | 14.5 ng/µL
|-
|-
-
|'''Total volume (e.g. 15,20,25,30 µl)'''|| align="right" | ||  align="right" | 10 ||  align="right" | 10
+
| align="left" | P310
 +
| align="left" | 220 mg
 +
| align="left" | 20 µL
 +
| align="left" | 2.4 ng/µL
|}
|}
 +
<br />
 +
<b>Comment:</b> The DNA concentrations were very bad, which could be due to the fact, that the 6xHis_middlelinker construct's size is just 86 bp and the affibody's size is just 210 bp...
<br />
<br />
-
*Transformation:
 
-
The transformation was done following the standard protocol using XL1B cells.
+
<p style="font-size:15px; font-weight: bold; color: #ff00ff;">Ligation</p>
-
<br/>
+
<br />
 +
<b>1. pCerulean_6xHis_middlelinker</b>
 +
{| border="1"
 +
| align="left" |  ||align="left"| '''6xHis_middlelinker''' ||align="left"| '''pCerulean'''
 +
|-
 +
| align="left" | '''Volume/µl''' ||align="left"| 2.57 ||align="left"| 6.43
 +
|}
<br/>
<br/>
 +
<b>2. pCerulean_Zegfr:1907</b>
 +
{| border="1"
 +
| align="left" |  ||align="left"| '''Zegfr:1907''' ||align="left"| '''pCerulean'''
 +
|-
 +
| align="left" | '''Volume/µl''' ||align="left"| 2.72 ||align="left"| 6.28
 +
|}
 +
<br />
-
====<p style="font-size:15px; background-color:#ff00ff;"><b>Strategy for VP2 N-terminal fusion and VP1 insertion of targeting moelcules</b></p>====
+
<p style="font-size:15px; font-weight: bold; color: #ff00ff;">Trafo</p>
-
 
+
-
Investigator: <b>Hanna</B>
+
<br />
<br />
-
<p><b>Comment:</b> In order to target the EGFR receptor of tumor cells via Affibody or Darpin (antibody fragment is also possible!!!), or in order to expose e.g. His-Tags on the virus surface, two approaches have been figured out. <br/>
+
Trafo was performed following the standard protocol. Cells: XL1b (60 µL), DNA amount per sample: 3 µL; cells were plated onto Kanamycin-plates.
<br/>
<br/>
-
[[File:Freiburg10_N-terminalFusion_Strategy.JPG]]
+
 
 +
====<p style="font-size:15px; background-color:#66bbff;"><b>PCR of SV 40 terminator</b></p>====
 +
<b>Investigator: Bea</b>
<br/>
<br/>
-
[[File:Freiburg10_N-terminalFusion_Strategy2.JPG]]
+
<p style="color:#66bbff;"><b>Comment:</b> In order to obtain the SV40 poly adenylation site and clone it into the iGEM standard plasmid pSB1C3 a PCR needs to be performed with the pEGFP-C1 which contains the SV40 terminator.</p>
-
[[File:Freiburg10 N-terminalFusion Strategy3.JPG]]
+
<br/>
<br/>
 +
<ul>
 +
<li>Plasmid used for PCR: pEGFP-C1 (P230)</li>
 +
<li>1:1000 dilution of P230 has been prepared</li>
 +
<li>Plasmid used as vector: pSB1C3_CFP1 (P51.2)</li>
 +
</ul>
 +
<br/>
 +
<b>PCR (was performed following the standard protocol)</b>
 +
<br>
-
 
-
===='''pSB1C3_Rep52 (P202) sent for sequencing'''====
 
-
 
-
'''Investigator: Chris L.'''<br>
 
-
 
-
Primer used: Reverse Primer VR2
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;">'''Repetition: Biobrick production of Rep 68, 78 and AAP'''</p>====
 
-
 
-
'''Investigator: Stefan'''<br>
 
-
 
-
 
-
Aim of the experiment:
 
-
We want to produce biobricks from Rep 68, Rep 78 and the AAP.
 
-
 
-
*Plasmids used as template:
 
-
Rep_68_ex (p119):  c = 470,6 ng/µl <br>
 
-
Rep_78_(p122):      c = 566,8 ng/µl <br>
 
-
pAAV-RC containing AAP ORF (p50):    c = 378,5 ng/µl <br>
 
-
 
-
 
-
 
-
*Primer used:
 
-
For Rep_68_ex: Praefix_68_78_ex & Suffix_40_68_ex<br>
 
-
For Rep_78_ex: Praefix_68_78_ex & Suffix_52_78_ex<br>
 
-
For AAP_ex: Praefix_AAP_ex & Suffix_AAP_ex
 
-
 
-
<br>
 
-
*'''PCR:
 
-
(was performed following the standard protocol)
 
-
<br>
 
{| border="1"
{| border="1"
-
| '''Ingredients''' || align="right" |'''Volume / µl''' || align="right" |'''Rep68'''|| align="right" |'''Rep78''' || align="right" |'''AAP'''
+
| '''Ingredients''' || align="right" |'''Volume / µl'''
|-
|-
-
| 5X Phusion HF buffer ||  align="right" |10 ||  align="right" | ||  align="right" | ||  align="right" |
+
| 5X Phusion HF buffer ||  align="right" |10  
|-
|-
-
| 10 mM dNTP mix||  align="right" |1||  align="right" |  ||  align="right" | ||  align="right" |
+
| 10 mM dNTP mix||  align="right" |1
|-
|-
-
| forward primer: ||  align="right" |2,5||  align="right" | ||  align="right" | ||  align="right" |
+
| forward primer: O160 ||  align="right" |2,5
|-
|-
-
| reverse primer: ||  align="right" |2,5||  align="right" | ||  align="right" | ||  align="right" |
+
| reverse primer: O161||  align="right" |2,5
|-
|-
-
| DNA Template||  align="right" |***||  align="right" |2,5 µl ||  align="right" |2 µl||  align="right" |3 µl
+
| DNA Template||  align="right" |3
|-
|-
-
| DMSO (2%)||  align="right" | ||  align="right" | - ||  align="right" | - ||  align="right" | 1 µl
+
|- DMSO ||  align="right" |-
|-
|-
-
| Phusion Polymerase||  align="right" |0,5||  align="right" | ||  align="right" | ||  align="right" |
+
| Phusion Polymerase||  align="right" |0,5
|-
|-
-
|H<sub>2</sub>O||  align="right" |*** ||  align="right" | 31 µl||  align="right" | 31,5 µl||  align="right" |29,5 µl
+
|H<sub>2</sub>O||  align="right" |30,5  
|-
|-
-
|Total volume||  align="right" |50
+
|'''Total volume'''||  align="right" |'''50'''
|}
|}
<br>
<br>
-
PCR program:
+
<b>PCR program:</b>
{| border="1"
{| border="1"
-
|'''PCR Program'''|| align="right" |'''temperature/ °C'''|| align="right" |'''Time''' || align="right" |'''Rep68'''|| align="right" |'''Rep78'''|| align="right" |'''AAP'''
+
|'''PCR Program'''|| align="right" |'''temperature/ °C'''|| align="right" |'''Time'''  
|-
|-
-
|1||  align="right" |98 ||  align="right" |1min||  align="right" | ||  align="right" | ||  align="right" |
+
|1||  align="right" |98 ||  align="right" |1min
|-
|-
-
|2||  align="right" |98 ||  align="right" |15s||  align="right" | ||  align="right" | ||  align="right" |
+
|2||  align="right" |98 ||  align="right" |15s
|-
|-
-
|3||  align="right" |*** ||  align="right" |25s||  align="right" |63°C||  align="right" |62°C||  align="right" |64°C
+
|8x||  align="right" |59 ||  align="right" |25s
|-
|-
-
|4 (step 2-4 8x) ||  align="right" |72||  align="right" |***||  align="right" |24s ||  align="right" |27s ||  align="right" | 10s
+
|3 ||  align="right" |72||  align="right" |7s
|-
|-
-
|5 ||  align="right" |98||  align="right" | 15s||  align="right" | ||  align="right" | ||  align="right" |
+
|4 ||  align="right" |98||  align="right" | 15s
|-
|-
-
|6||  align="right" |***||  align="right" |25s|| align="right" |68°C||  align="right" |64°C||  align="right" |68°C
+
|17x||  align="right" |67||  align="right" |25s
|-
|-
-
|7||  align="right" |72||  align="right" |***||  align="right" |24s||  align="right" |27s||  align="right" |10s
+
|5||  align="right" |72||  align="right" |7s
|-
|-
-
|8 (step 6-8 17x)||  align="right" |72||  align="right" |5min||  align="right" | ||  align="right" | ||  align="right" |
+
|6x||  align="right" |72||  align="right" |5min
|-
|-
-
|Hold||  align="right" |4||  align="right" |  ||  align="right" | ||  align="right" | ||  align="right" |
+
|Hold||  align="right" |4||  align="right" |   
|}
|}
-
<br>
+
After the PCR was performed, the sample was loaded on a 1.5% agarose gel and run for 30 minutes. As it can be seen in the picture below the expected size of the PCR product (260bp) can be confirmed. The lower band corresponds to the added primers and does not seem to be an additional band.
-
Used agarose gel:
+
<gallery widths=300px heights=300px>
-
0,5 g Agarose,50 ml TBE (1%), 3 µl GELRED , at 115 Volt, running time:45 minutes
+
Image:Freiburg10 PCR product SV40.jpg
 +
</gallery>
-
[[File:Freiburg10 68 78 AAP.jpg|500px|]]
+
<br>
-
 
+
For cloning the obtained fragment into the pSB1C3 standard vector, the construct need to be digested with the same restriction enzymes as the PCR product will be cut with. In this case the primers were designed with XbaI overhang for the prefix, and with SpeI-NotI-PstI overhang for the suffix. Therefore the vector will be cut with XbaI and PstI. The sample were incubated at 37 C for approx. 1.5 h
-
 
+
-
<p style="font-size:15px; font-weight: bold; color: blue;">Gel extraction</p>
+
<br />
<br />
-
Gel extraction was perfomed according to protocol.
+
<ul>
 +
<li>Plasmid used: pSB1C3_CFP (P51.2) c=150ng/µL</li>
 +
<li>Add BSA</li>
 +
<li>XbaI and PstI were used</li>
 +
</ul>
 +
<br/>
 +
<b>Digestion of vector:</b>
-
*Digestion of PCR products and vector:
 
-
 
-
<br />
 
{| border="1"
{| border="1"
-
| '''components'''  || align="right" |'''PCR products /µl''' || align="right" |'''vector /µl'''
+
| align="left" | '''Components''' ||align="left"| <b>v<sub>pSB1C3_CFP</sub></b>/µL
|-
|-
-
| DNA  ||  align="right" |29 || align="right" |11
+
| align="left" | DNA  ||align="left"| 8
|-
|-
-
| BSA (10x) ||  align="right" |4 || align="right" | 2
+
| align="left" | BSA (100x) ||align="left"| 2
|-
|-
-
| Buffer 4 (10x)||  align="right" |4 || align="right" |2  
+
| align="left" | Buffer no. 4 (10x) ||align="left"| 2
|-
|-
-
|Enzyme '''XbaI ||  align="right" |1 || align="right" |1  
+
| align="left" | Enzyme 1 XbaI ||align="left"| 1
|-
|-
-
|Enzyme '''SpeI ||  align="right" |1 || align="right" |1  
+
| align="left" | Enzyme 2 AgeI HF ||align="left"| 1
|-
|-
-
|H2O||  align="right" |1 || align="right" |3
+
| align="left" | H<sub>2</sub>O ||align="left"| 6
|-
|-
-
|'''Total volume (e.g. 15,20,25,30 µl)'''|| align="right" | 40|| align="right" |20
+
| align="left" | '''Total volume''' ||align="left"| <b>20</b>  
|}
|}
<br />
<br />
-
<p style="font-size:13px; color:#68bbff;">'''''Comments''''': Digestion was done with XbaI and SpeI, it has to be checked if the inserts are cloned into the vector in the right orientation. </p> <br>
+
<ul>
-
 
+
<li>Incubation of the sample at 37°C</li>
-
*Purification of Rep68, 78 and AAP:
+
<li>Incubation for 120 minutes</li>
-
For the purification 200 µl of buffer PBI was used.
+
</ul>
-
 
+
-
c(Rep68)= 14,3 ng/µl <br />
+
-
c(Rep78)= 12,0 ng/µl <br />
+
-
c(AAP)= 25,1 ng/µl
+
-
 
+
<br />
<br />
-
 
+
After the PCR has been cut out of the gel and purified with the Qiagen Gel Extraction Kit obtaining a concentration of 45 ng/µl measured with the Nanodrop, the complete PCR product volume of 29 µL was digested with XbaI and PstI for 2 hours.<br />
-
*Gelextraction of pSB1C3_RFC25_CFP:
+
-
0,5 g Agarose,50 ml TBE (1%), 3 µl GELRED , at 115 Volt, running time:50 minutes <br />
+
-
4µl loading dye (6x) for the sample, Marker: GeneRuler ladder mix (Fermentas)
+
-
<br />
+
-
<br />
+
-
<br />
+
-
<br />
+
-
[[File:Freiburg10 pSB1C3 CFP 130810.jpg|500px|]]
+
-
 
+
-
 
+
-
<br />
+
-
 
+
-
 
+
-
c(pSB1C3)= 6,9 ng/µl <br />
+
-
 
+
-
 
+
-
<br />
+
-
 
+
-
*T4 ligation of PCR products and vector:
+
-
 
+
-
For the Ligation 1µl buffer (10x) and 1µl T4 ligase were used.
+
<br />
<br />
 +
<b>Digestion of the PCR product SV40</b>
{| border="1"
{| border="1"
-
| ''' '''  || align="right" |'''vector /µl''' || align="right" |'''insert /µl'''
+
| align="left" | '''Components''' ||align="left"| <b>v<sub>PCR product</sub></b>/µL
|-
|-
-
| pSB1C3 + Rep68 || align="right" |3,78 || align="right" |4,24
+
| align="left" | DNA  ||align="left"| 29,0 µl
|-
|-
-
| pSB1C32 + Rep78 ||  align="right" |3,13 || align="right" |4,87
+
| align="left" | BSA (10x) ||align="left"| 4,0 µl
|-
|-
-
| pSB1C3 + AAP ||  align="right" |6,42 || align="right" |1,58
+
| align="left" | Buffer no. 4 (10x) ||align="left"| 4,0 µl
|-
|-
-
 
+
| align="left" | Enzyme 1 XbaI ||align="left"| 1,0 µl
 +
|-
 +
| align="left" | Enzyme 2 AgeI HF ||align="left"| 1,0 µl
 +
|-
 +
| align="left" | H<sub>2</sub>O ||align="left"| 1,0 µl
 +
|-
 +
| align="left" | '''Total volume''' ||align="left"| <b>40</b> 
|}
|}
 +
<ul>
 +
<li>Incubation of the sample at 37°C</li>
 +
<li>Incubation for 120 minutes</li>
 +
</ul>
<br />
<br />
 +
The digested PCR product SV40 was purifed with the PC Purification Kit and concentration was measured.
 +
<ul>
 +
<li><b>c<sub>SV40</sub>= 31,5 ng/µl</b></li>
 +
<li><b>c<sub>pSB1C3</sub>= 7,74 ng/µL</b></li>
 +
</ul>
 +
For T4 ligation of the two fragments, the needed volumes of insert (SV40) and vector (pSB1C3) were calculated with the labtools program. The total volume DNA (vector/insert) mix will be 8 µL.
 +
<ul>
 +
<li><b>v<sub>SV40</sub>= 0,67 µL</b></li>
 +
<li><b>v<sub>pSB1C3</sub>= 7,33 µL</b></li>
 +
<li><b>v<sub>T4 buffer</sub>= 1 µL</b></li>
 +
<li><b>v<sub>t4 ligase</sub>= 1 µL</b></li>
 +
</ul>
 +
The ligation reaction was carried out for 40 minutes at room temperature. The ligated plasmid was transformed into <b>XL1-Blue cells</b> and plated on agar plates containing the appropriate amount of <b>chloramphenicol</b>.
-
*Transformation:
+
====<p style="font-size:15px; background-color:#33FF88;"><b>Cloning of NLS into pCerulean_VP1up</b></p>====
-
 
+
Investigator: Anissa
-
The transformation was done following the standard protocol using XL1b cells.
+
-
 
+
-
=== 89. labday 14.08.2010===
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Sequence analysis of pSB1C3_SDM_SspI_BLA</b></p>====
+
-
Investigator: <b>Patrick, Bea </b>
+
-
<br />
+
-
<p style="font-size:13px; color:#003399;"><b>Comments</b>: Sequence analysis of three clones revealed that all sequences contain some mutations in the regions where the primer annealed. This was due to the extreme primer lentgh. Primers that long should be ordered as HPLC purified in order to avoid mutations in the primer sequence.</p>
+
-
<ol>
+
-
<li>Clone 1: Mutation in the SalI site of the loop insertion site in the Viral Brick MCS lead to the removal of the recognition site. CANNOT be used for the loop insertions!!!!</li>
+
-
<li>Clone 2:  Mutation in the region of the primer upstream of the loop insertion sites SalI and PvuII.</li>
+
-
<li>Clone 3: Mutation in the region of the primer downstream of the loop insertion sites SalI and PvuII.</li>
+
-
</ol>
+
-
<br />
+
-
<gallery widths=450px heights=300px perrow=2 caption="Sequencing results of pSB1C3_SDM of SspI_BLA clone 2 and pSB1C3_SDM of SspI_BLA clone 3">
+
-
Image:Freiburg10 Sequence analysis of pSB1C3 SDM SspI BLA clone2.jpg|Clone 2
+
-
Image:Freiburg10 Sequence analysis of pSB1C3 SDM SspI BLA clone3.jpg|Clone 3
+
-
</gallery>
+
-
<br />
+
-
<p style="font-size:13px; color:#33cc66;"><b>Next steps: </b> Two additional clones will be picked and sent for sequencing (clones 4 and 5). In order to obtain a construct without any mutations, clone 2 and clone 3 couldbe cloned together. Each clone will be digested with SalI and BamHI. Clone 2 will be the "insert", clone 3 is going to be the "vector". Therefore the fragments will (hopefully) not contain anymore mutations in the relevant sequence. </p>
+
-
 
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Mini Preps of PMA_RepCap vector and pSB1C3_left ITR_CMV_betaglobin_mVenus</b></p>====
+
<br/>
<br/>
-
'''Investigator: Chris L., Bea'''
+
<p style="color:#33FF88;"><b>Comment:</b> NLS was already 2 times cut of the pSB1C3_NLS and purified by Patrick (NLS 1: of the 29.08.10, NLS 2: of the 30.08.10) . Now, pCerulean_VP1up will be digested and NLS will be ligated into it.</p>
-
<br>
+
-
<p style="font-size:13px; color:#003399;"><b>Comments</b>:new mini-preps of the glycerol-stocks of pSB1C3_leftITR_CMV_betaglobin_mVenus clone 1 (=B179), pSB1C3_leftITR_CMV_betaglobin_mVenus clone 2 (=B180), pSB1C3_leftITR_CMV_betaglobin_mVenus clone 3 (=B181), pMA_RepCap Vector_SDM_InsPvuII clone 1, (=B182), pMA_RepCap Vector_SDM_InsPvuII clone 2 (=B183) and pMA_RepCap Vector_SDM_InsPvuII clone 3 (=B184)</p>
+
<br/>
<br/>
-
<b>Comment: The plamsid numbers in the picture (P179-P184) correspond to the glycerol stocks B179-B184. The corresponding plasmid numbers are: P208-P213. </b>
+
<ul>
-
<br />
+
<li>Digestion of pCerulean_VP1up (p303)
-
'''Nanodrop'''
+
<br/><br/>
-
* pSB1C3_leftITR_CMV_betaglobin_mVenus clone 1: 256,64 ng/µl '''B179'''
+
-
* pSB1C3_leftITR_CMV_betaglobin_mVenus clone 2: 274,86 ng/µl '''B180'''
+
-
* pSB1C3_leftITR_CMV_betaglobin_mVenus clone 3: 217,20 ng/µl '''B181'''
+
-
* pMA_RepCap Vector_SDM_InsPvuII clone 1: 253,71 ng/µl '''B182''' 
+
-
* pMA_RepCap Vector_SDM_InsPvuII clone 2: 195,64 ng/µl '''B183'''
+
-
* pMA_RepCap Vector_SDM_InsPvuII clone 3: 144,61 ng/µl '''B184'''
+
-
 
+
-
<br>
+
-
<br>
+
-
'''Test digestion''' 800ng DNA
+
{| border="1"
{| border="1"
-
| components  || align="right" |B179 /µl || align="right" |B180 /µl || align="right" |B181 /µl || align="right" |B182 /µl|| align="right" |B183 /µl || align="right" |B184 /µl|| align="right" |pMA_RepCap Vector /µl
+
| components  || align="right" |Vector  
|-
|-
-
| DNA  ||  align="right" | 3,1||  align="right" |2,9||  align="right" |3,7||  align="right" |3,1||  align="right" |4||  align="right" |5,5||  align="right" | 2,4
+
| DNA  ||  align="right" |2,6
|-
|-
-
| BSA (10x) ||  align="right" | 2||  align="right" |2||  align="right" |2||  align="right" |2||  align="right" |2||  align="right" |2||  align="right" |2
+
| BSA (10x) ||  align="right" |1,5
|-
|-
-
| Buffer 4 (10x)||  align="right" | 2||  align="right" |2||  align="right" |2||  align="right" |2||  align="right" |2||  align="right" |2||  align="right" |2
+
| Buffer 4 (10x)||  align="right" |1,5
|-
|-
-
| Enzyme 1: NgoMIV ||  align="right" | 0,5||  align="right" |0,5||  align="right" |0,5||  align="right" |0||  align="right" |0||  align="right" |0 ||  align="right" |0
+
|Enzyme PstI ||  align="right" |1
|-
|-
-
| Enzyme 2: EcoRI || align="right" | 0,5||  align="right" |0,5||  align="right" |0,5||  align="right" |0||  align="right" |0||  align="right" |0||  align="right" |0
+
|Enzyme AgeI ||  align="right" |1
|-
|-
-
| Enzyme 2: PvuII ||  align="right" | 0||  align="right" |0||  align="right" |0 ||  align="right" |0,5||  align="right" |0,5||  align="right" |0,5||  align="right" |0,5
+
|H<sub>2</sub>O||  align="right" |7,4
|-
|-
-
|H<sub>2</sub>O||  align="right" | 11,9||  align="right" |12,1||  align="right" |11,3||  align="right" |12,4||  align="right" |11,5||  align="right" |10||  align="right" | 13,1
+
|'''Total volume '''||  align="right" |15
-
|-
+
-
|'''Total volume (e.g. 15,20,25,30 µl)'''||  align="right" | '''20'''||  align="right" | '''20'''||  align="right" | '''20'''||  align="right" | '''20'''||  align="right" | '''20'''||  align="right" | '''20'''||  align="right" | '''20'''
+
|}
|}
<br>
<br>
-
Incubation time: 45 minutes, Incubation temperature: 37°
+
</li>
-
<br>
+
<li>Gel:
-
<br>
+
<br/>
-
<br>
+
A 1% gel was made and the sample was running 30 minutes.<br/>
-
0,5 g Agarose, 50ml x TAE, 3µl GELRED, at 115 Volt, running time: 45 minutes
+
[[image:Anissa310810.png|300px]]<br/>
 +
Afterwards a gelextraction according the qiagen-protocol was performed.<br/><br/>
 +
</li>
 +
<li>Ligation: <br/>
 +
{| border="1"
 +
| components  || align="right" |Volume /µl || align="right" |concentration /ng/µl
 +
|-
 +
| NLS 2 insert of 30.08.10  ||  align="right" |1,51 || align="right" |3
 +
|-
 +
| NLS 1 insert of 29.08.10||  align="right" |1,51|| align="right" |3
 +
|-
 +
| pCerulean_VP1up||  align="right" |6,49 || align="right" |29
 +
|}<br/>
 +
</li><br/>
 +
<li>Transfomation: was performed into XL1 blue on a kanamycin plate according the standard protocol.
 +
</li>
 +
</ul>
-
<br />
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273)</b></p>====
-
<b>Results of test digestion</b>
+
-
[[File:Freiburg10 Test digestion of pSB1C3 Bba fourparts SDM pMA RC insert 14 08.jpg|500px]]
+
Investigator: Patrick <br>
-
<br />
+
P276 digestion (207 ng/µl): 14 µl DNA sample, 2 µl Buffer 4, 2 µl BSA, 1 µl XbaI, 1 µl PstI-HF. <br>
-
<br />
+
P273 digestion (419 ng/µl): 4 µl DNA sample, 1 µl Buffer 4, 1 µl BSA, 1 µl XbaI, 1 µl PstI-HF. <br>
-
<br />
+
Digestion time: 1 h 45 min.
-
<b>Results</b>: Two (three) different approaches can be seen on the agarose gel.
+
-
<ol>
+
-
<li style="font-size:13px; color:#66bbff;">P178-P181 (blue): The plasmid pSB1C3_leftITR_CMV_betaglobin_mVenus was digested with NgoMIV and EcoRI. Expected bands were: <b> 2873bp and 1343 </b>. As it can be seen in the picture, the two fragments ran too high (~300bp). As it is almost normal for the pSB1C3 plasmid, we should figure out why this is always the problem for this vector.</li>
+
-
<li style="font-size:13px; color:#cc3333;">P182-P183 (red): The plasmid pMA_RepCap_SDM_insPvuII was digested with PvuII as well as the control vetor pMA_RepCap where no site-directed mutagenesis was performed in order to see the difference. As it can be seen in the picture above, the expected band sizes of <b>263bp,1384bp and 2133bp</b> correspond to the bands detected in the gel. The control vector instead reveals only two bands detectable. Therefore site-directed mutagenesis was successfully performed.</li>
+
-
<li style="font-size:13px; color:#33cc33;">P177 (green): pSB1C3_hTERT. For further details see extra part below (Test digestion of pSB1C3_hTERT.</li>
+
-
</ol>
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-Prep of pSB1C3_leftITR_CMV_mVenus</b></p>====
+
Expected size of the fragments: <br>
-
<b>Investigator: Stefan</b><br>
+
P276: 786 & 2058 bp <br>
 +
P273: 756 & 3938 bp <br>
-
Glycerol stocks were prepared:
+
[[image:Cartoons48.jpg|thumb|right| My dear friend, where is your initial comment? Nice gel :)
-
<li>B177 = pSB1C3_leftITR_CMV_mVenus clone 1
+
Yours sincerely, Hanna :)]]
-
<li>B178 = pSB1C3_leftITR_CMV_mVenus clone 2
+
see: http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/August_2010#Strategy_for_VP2_N-terminal_fusion_and_VP1_insertion_of_targeting_moelcules
 +
<br/>
 +
<font color="#ff00ff">Haha, good idea! :) </font><br/>
 +
[[File:Freiburg10_3108pat2.JPG|500px]]
-
Mini-Prep following the standard protokol
+
Gelextraction:
-
<br />
+
Obviously there was something wrong with the gel or the sample. The marked bands were cut out and the gelextraction was performed acording to he standard protocol, yielding the following concentrations:<br>
-
<li>P206 = pSB1C3_leftITR_CMV_mVenus clone 1 = 277,9 ng/µl
+
P276 up: 12,6 ng/µl  <br>
-
<li>P207 = pSB1C3_leftITR_CMV_mVenus clone 2 = 260,3 ng/µl
+
P276 down: 9,9 ng/µl <br>
 +
P273: 37,7 ng/µl <br>
 +
 
 +
All of them were polluted.
 +
 
 +
There were two bands at a level where no bands should be. P 276 looks also strange because accidentally 6,5 µl 1kb GeneRuler were pipetted into the sample. The expected bands are all blurred. Even the expected 756 bp fragment of P273 is not at the level it should be. To ensure i dont throw away the insert i cut out both bands from P276. Now tomorrow there will be a ligation and a transformation with two samples instead of one
 +
 
 +
 
 +
<br/>
-
<br />
+
====<p style="font-size:15px; background-color:#ff00ff;"><b>Sequencing results of pSB1C3_6xHis_middlelinker and pSB1C3_6xHis_mVenus</b></p>====
-
<br>
+
Investigator: Hanna
-
<br>
+
<br/>
-
<b>Test digestion:</b>
+
-
or test digestion, pSB1C3_leftITR_CMV (P188) was digested as well. <br />
+
 +
<p><b>1st Comment:</b> Sequencing of pSB1C3_6xHis_middlelinker looked good:</p>
 +
<br/>
 +
[[File:Freiburg10 6xHis middlelinker.JPG|600px]]
 +
<br/>
 +
<p><b>2nd Comment:</b> Sequencing of mVenus looked good. The His-Tag delivered 2 additional bp and the suffix 1 transition (see picture). This could be due to bad sequencing qualities at this region... but needs to be verified!</p>
 +
[[File:Freiburg10 6xHis mVenus.JPG|700px]]
 +
 +
 +
====<p style="font-size:15px; background-color:#66bbff;"><b>Contiuation: Repetition of subcloning Cap-insert into pAAV_RC</b></p>====
 +
<b>Investigator: Stefan</b>
 +
 +
<b>Comments:</b> Overnight digestion does not look good, therefore a new approach will be performed today using different concentrations of spermidine (0,5mM, 2,5mM and 10mM) because in Oller et al (Biochemistry, 1991) it was suggested that this improves digestion with BspMI.
 +
 +
A two-step digestion was performed: <br />
 +
<br />
 +
'''1st step'''
{| border="1"
{| border="1"
-
| '''Ingredients''' || align="right" |'''clone 1/µl'''|| align="right" |'''clone 2/µl''' || align="right" |'''P188/µl'''
+
| || align="right" |'''P211 / µl''' ||'''P250 / µl'''
|-
|-
-
| DNA: ||  align="right" |3,5 || align="right" |3,5 ||  align="right" |4
+
|DNA||  align="right" |8,5  ||  align="right" | 2                             
|-
|-
-
| BSA (10x): ||  align="right" |2  ||  align="right" |2 ||  align="right" |2
+
|buffer 3||  align="right" |4,6 ||  align="right" | 5
|-
|-
-
| Buffer 4: ||  align="right" |2 || align="right" |2 ||  align="right" |2
+
| Enzyme BsiWI||  align="right" |1 ||  align="right" | 1
|-
|-
-
| Enzyme 1 EcoRI: ||  align="right" |0,5 ||  align="right" |0,5 ||  align="right" |0,5
+
|H<sub>2</sub>O||  align="right" |32,4 ||  align="right" | 38
|-
|-
-
| Enzyme 2 AgeI||  align="right" |0,5 ||  align="right" |0,5|| align="right" |0,5
+
|total volume||  align="right" |46 ||  align="right" |46
 +
|}
 +
<br />
 +
*Incubation: 70 minutes; 55°C<br>
 +
'''2nd step'''
 +
{| border="1"
 +
| || align="right" |'''P211 / µl''' ||'''P250 / µl'''
|-
|-
-
|H<sub>2</sub>O||  align="right" | 6,5|| align="right" | 6,5||  align="right" |6
+
|Mix obtained from step 1 ||  align="right" |46 ||  align="right" | 46                             
|-
|-
-
 
+
| Enzyme BspMI||  align="right" |4  ||  align="right" | 4
 +
|-
 +
|total volume||  align="right" |50 ||  align="right" |50
|}
|}
-
 
<br />
<br />
-
<ul>
+
*Incubation: 2,5h ; 37°C<br>
-
All samples were loaded on a 1% agarose gel:
+
'''Agarosegel'''
-
<li>GeneRuler Ladder Mix (7µl)</li>
+
<br />
-
<li>P206 = pSB1C3_leftITR_CMV_mVenus clone 1 </li>
+
0.4 g Agarose, 50 ml TAE (0,8 %), 3 µL GELRED, at 115 Volt
-
<li>P207 = pSB1C3_leftITR_CMV_mVenus clone 2 </li>
+
<br />
-
<li>P188 = pSB1C3_leftITR_CMV</li>
+
<br />
 +
 
-
</ul>
 
<br />
<br />
-
[[File:Freiburg10 test digestion leftITR CMV mVenus 140810.jpg|300px]]
+
[[File:Freiburg10 P250 without Spermidine.png|300px|left|thumb|]]
 +
[[File:Freiburg10 P250 with spermidine.png|500px|right|thumb|]]
<br />
<br />
<br />
<br />
<br />
<br />
-
comment: Through digestion, fragments would have similar sizes. Therefore, a new digestion will be performed tomorrow.
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Test digestion of pSB1C3_pHTERT</b></p>====
 
-
Investigator: <b>Bea </b>
 
<br />
<br />
-
<p style="font-size:13px; color:#003399;"><b>Comments</b>: pSB1C3_hTERT was sent for sequencing and revealed that the PCR and the inserting of the PCR product : promoter for hTERT was sucessfully performed. But the sequencing results showed a new AgeI restriciton site in the plasmid backbone. We could not see if the new recognition site was due to the bad sequence read or that there is really a new AgeI site. Therefore, a test digestion will be perfomed with PstI and AgeI in order to figure our wheter AgeI is present in the backbone or not.</p>
 
<br />
<br />
-
<p style="font-size:13px;"><b>Sequence analysis of pSB1C3_hTERT: </b></p>
 
-
<gallery widths=450px heights=300px perrow=2 caption="Sequencing results of pSB1C3_hTERT">
 
-
Image:Freiburg10 Sequence analysis of pSB1C3 hTERT suffix.jpg
 
-
Image:Freiburg10 Sequence analysis of pSB1C3 hTERT CAT mutations.jpg
 
-
</gallery>
 
<br />
<br />
<br />
<br />
-
<b>First the sequenced plasmid P177 was digested:</b>
 
<br />
<br />
-
<table border=1 cellpadding=1 cellspacing=1 width=284 style='border-collapse:
 
-
collapse;table-layout:fixed;width:214pt'>
 
-
<tr height=30 style='mso-height-source:userset;height:22.5pt'>
 
-
  <td height=30 class=xl6312733 width=142 style='height:22.5pt;width:107pt'>&nbsp;</td>
 
-
  <td class=xl6412733 width=142 style='border-left:none;width:107pt'>P177/µL</td>
 
-
</tr>
 
-
<tr height=30 style='mso-height-source:userset;height:22.5pt'>
 
-
 
-
  <td height=30 class=xl6512733 width=142 style='height:22.5pt;border-top:none;
 
-
  width:107pt'>DNA</td>
 
-
  <td class=xl6612733 style='border-top:none;border-left:none'>3</td>
 
-
</tr>
 
-
<tr height=30 style='mso-height-source:userset;height:22.5pt'>
 
-
  <td height=30 class=xl6512733 width=142 style='height:22.5pt;border-top:none;
 
-
  width:107pt'>BSA (10x)</td>
 
-
  <td class=xl6612733 style='border-top:none;border-left:none'>1,5</td>
 
-
</tr>
 
-
 
-
<tr height=30 style='mso-height-source:userset;height:22.5pt'>
 
-
  <td height=30 class=xl6512733 width=142 style='height:22.5pt;border-top:none;
 
-
  width:107pt'>Buffer 4<span style='mso-spacerun:yes'>  </span>(10x)</td>
 
-
  <td class=xl6612733 style='border-top:none;border-left:none'>1,5</td>
 
-
</tr>
 
-
<tr height=30 style='mso-height-source:userset;height:22.5pt'>
 
-
  <td height=30 class=xl6512733 width=142 style='height:22.5pt;border-top:none;
 
-
  width:107pt'>Enzyme PstI</td>
 
-
 
-
  <td class=xl6612733 style='border-top:none;border-left:none'>0,5</td>
 
-
</tr>
 
-
<tr height=30 style='mso-height-source:userset;height:22.5pt'>
 
-
  <td height=30 class=xl6512733 width=142 style='height:22.5pt;border-top:none;
 
-
  width:107pt'>Enzyme AgeI</td>
 
-
  <td class=xl6612733 style='border-top:none;border-left:none'>0,5</td>
 
-
</tr>
 
-
<tr height=30 style='mso-height-source:userset;height:22.5pt'>
 
-
 
-
  <td height=30 class=xl6512733 width=142 style='height:22.5pt;border-top:none;
 
-
  width:107pt'>H2O</td>
 
-
  <td>8</td>
 
-
</tr>
 
-
<tr height=30;height:22.5pt'>
 
-
  <td height=30  width=142 style='height:22.5pt;
 
-
  width:107pt'><b>Total volume</b></td>
 
-
  <td><b>15</b></td>
 
-
</tr>
 
-
 
-
<tr height=0 style='display:none'>
 
-
  <td width=142 style='width:107pt'></td>
 
-
  <td width=142 style='width:107pt'></td>
 
-
</tr>
 
-
</table>
 
<br />
<br />
-
<ul>
 
-
<li>Incubation time: 45minutes</li>
 
-
<li>Incubation tempereature: 37°C</li>
 
-
</ul>
 
-
After the incubation,the samples were loaded on the 1% agarose gel, and run at 110 V for 45 minutes.
 
<br />
<br />
<br />
<br />
-
<b>Result of the test digestion:</b>
 
<br />
<br />
-
<gallery widths=400px heights=300px caption="Test digestion of pSB1C3_hTERT">
 
-
Image: Freiburg10 Testdigestion pSB1C3 hTERT 14 08.jpg
 
-
</gallery>
 
<br />
<br />
-
<p style="font-size:13px; color:#33cc33;"><b>Results:</b> As it can be seen in the sequencing results of pSB1C3_hTERT there could have been an additional AgeI site in the vector. If AgeI would have been in the vector, two fragments have been expected by digetsing the plasmid with AgeI and PstI. As it can be seen in the agarose gel, lane 11 (labeled with the green P177) only reveals one lane instead of two. Therefore it can be assumed that NO AgeI site is in the pSB1C3 backbone.</p>
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Picking clones ... </b></p>====
 
-
Investigator: Patrick
 
-
15 Clones were picked saturday night so that a mini-prep could be performed on sunday.
 
-
 
-
=== 90. labday 15.08.2010===
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Sequence analysis of pSB1C3_Rep52</b></p>====
 
-
<b>Investigator: Bea </b>
 
<br />
<br />
-
<p style="font-size:13px; color:#003399;"><b>Comments</b>: Clone 3 was sent for sequencing because this clone revealed the right band sizes of the test digestion performed friday. For more details see: [http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal#Mini-Prep_and_Test_digestion_of_Rep40_and_Rep52_of_the_cloning_experiment_.28.22Cloning_of_Rep40.2F52.22_11.08.2010_Investigator:_Anna.29 Test digestion] </p>
 
-
 
-
<gallery widths=700px heights=300px caption="Sequencing results of pSB1C3_Rep52 clone 3">
 
-
Image:Freiburg10 Sequence analysis of pSB1C3 Rep52 14 08 2010.JPG
 
-
</gallery>
 
<br />
<br />
-
<b>Results:</b> <p style="color:#00bbff;"> Sequence assembly showed that the suffix of the Rep protein 52 is ok and that PCR worked well. </p> <p style="color:#990000;">There can be seen that a silent mutation from AGG to AGA was inserted, but it will have no effect on the protein because codon usage in <i>H. sapiens </i>is nearly the same for both codons.</p>
 
-
The prefix could not be analysed because of bad sequence read quality. For further verification, another sequencing with the forward primer has to be performed.
 
<br />
<br />
-
<b>Next steps: Send plasmid for another sequencing round with forward primer in order to analyse the prefix. </b>
 
<br />
<br />
<br />
<br />
-
[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]<br />
 
-
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-Prep of pSB1C3_Rep68, pSB1C3_Rep78, pSB1C3_RepAAP, pSB1C3_Rep40, pSB1C3_Rep52</b></p>====
 
-
<b>Investigator: Stefan</b><br>
 
-
<ul>
 
-
<b>Glycerol stocks were prepared:</b>
 
-
<li>B185 = pSB1C3_Rep68 clone 1
 
-
<li>B186 = pSB1C3_Rep68 clone 2
 
-
<li>B187 = pSB1C3_Rep68 clone 3<br />
 
<br />
<br />
-
 
-
<li>B188 = pSB1C3_Rep78 clone 1
 
-
<li>B189 = pSB1C3_Rep78 clone 2
 
-
<li>B190 = pSB1C3_Rep78 clone 3<br />
 
<br />
<br />
-
 
-
<li>B191 = pSB1C3_AAP clone 1
 
-
<li>B192 = pSB1C3_AAP clone 2
 
-
<li>B193 = pSB1C3_AAP clone 3<br />
 
<br />
<br />
-
 
-
<li>B194 = pSB1C3_Rep40 clone 1
 
-
<li>B195 = pSB1C3_Rep40 clone 2<br />
 
<br />
<br />
-
 
-
<li>B196 = pSB1C3_Rep52 clone 1
 
-
<li>B197 = pSB1C3_Rep52 clone 2<br />
 
<br />
<br />
-
 
-
<li>B198 = pSB1C3_SDM_SspI_Bla14FM clone 4
 
-
<li>B199 = pSB1C3_SDM_SspI_Bla14FM clone 5<br />
 
<br />
<br />
<br />
<br />
<br />
<br />
-
 
-
 
-
<b>Mini-Prep following the standard protokol</b>
 
<br />
<br />
-
<li>P208 = pSB1C3_Rep68 clone 1 c=230,0
 
-
<li>P209 = pSB1C3_Rep68 clone 2 c=160,5
 
-
<li>P210 = pSB1C3_Rep68 clone 3 c=128,6 <br />
 
<br />
<br />
-
 
-
<li>P211 = pSB1C3_Rep78 clone 1 c=150,8
 
-
<li>P212 = pSB1C3_Rep78 clone 2 c=108,6
 
-
<li>P213 = pSB1C3_Rep78 clone 3 c=131,3<br />
 
<br />
<br />
-
 
-
<li>P214 = pSB1C3_AAP clone 1 c=142,1
 
-
<li>P215 = pSB1C3_AAP clone 2 c=148,3
 
-
<li>P216 = pSB1C3_AAP clone 3 c=163,0<br />
 
<br />
<br />
-
 
-
<li>P217 = pSB1C3_Rep40 clone 1 c=164,5
 
-
<li>P218 = pSB1C3_Rep40 clone 2 c=<145,1br />
 
<br />
<br />
-
<li>P219 = pSB1C3_Rep52 clone 1 c=156,0
+
'''Gel extraction'''
-
<li>P220 = pSB1C3_Rep52 clone 2 c=164,0<br />
+
<br />  
 +
Gel-Ex was performed according to protocol.<br />
 +
NanoDrop:
 +
*pMA_RepCap-insert: c= 7,42 ng/µl
 +
*pAAV_RC_ins-rep: c=12,9 ng/µl
<br />
<br />
-
<li>P221 = pSB1C3_SDM_SspI_Bla14FM clone 4 c=220,4
+
'''T4 Ligation'''
-
<li>P222 = pSB1C3_SDM_SspI_Bla14FM clone 5 c=252,2<br />
+
<br />  
-
</ul>
+
Volume insert: 3,43 µl<br>
 +
Volume vector: 4,57 µl<br>
 +
Ligation incubated for 50 minutes at room temperature.<br />
<br />
<br />
-
<br />
+
'''Transformation'''
-
<br />
+
Trafo was preformed according to standard protocol using XL1blue cells on plates containing Ampicilin.
-
<b>Test digestion:</b> <br />
+
-
No test digestion for pSB1C3_SDM_SspI_Bla14FM clone 4 and 5.<br />
+
-
For test digestion, pSB1C3_leftITR_CMV_mVenus (P206-207) and pSB1C3_leftITR_CMV (P188) was digested as well. <br />
+
-
Samples P208-220 were digested with XbaI and SpeI.<br />
+
-
Samples P207, P207 and P188 were digested with XbaI and NgoMIV.<br />
+
 +
====<p style="font-size:15px; background-color:#66bbff;"><b>2nd Repetition of test digestion of Loop insertion BioBricks</b></p>====
 +
<b>Investigator: Achim, Anna </b><br>
-
Gel used:<br />
+
<p style="font-size:13px; color:#003399;"><b>Comment</b>: First test digestion didn't work (see 31.08.10), this time a new approach was done with NotI. </p>
-
100 ml TAE (1x), 1 g agarose, 6 µl GELRED<br />
+
 
-
115 V, 50 minutes
+
<b>Test digestion:</b>
 +
<br>
{| border="1"
{| border="1"
-
| '''Ingredients''' || '''P208/µl'''|| |'''P209/µl''' || |'''P210/µl'''|| '''P211/µl'''|| |'''P212/µl''' || '''P213/µl'''|| |'''P214/µl''' || '''P215/µl'''|| |'''P216/µl''' || '''P217/µl'''|| |'''P218/µl''' || '''P219/µl'''|| |'''P220/µl''' || '''P206/µl'''|| |'''P207/µl''' || |'''P188/µl'''
+
| components ||Volume for each sample /µl  
|-
|-
-
| DNA: || |4||  |6 ||  |7  ||  |6 ||  |9 || |7 ||  |6 ||  |6 ||  |6||  |6 ||  |6  ||  |6 ||  |6 || |3,5 ||  |3,5 ||  |4
+
| DNA  || 10
|-
|-
-
| BSA (10x): || |2  ||  |2 ||  |2||  |2  ||  |2 ||  |2||  |2  ||  |2 ||  |2||  |2  ||  |2 ||  |2||  |2  ||  |2 ||  |2||  |2
+
| BSA (10x) ||1,5
|-
|-
-
| Buffer 4: || |2  ||  |2 ||  |2||  |2  ||  |2 ||  |2||  |2  ||  |2 ||  |2||  |2  ||  |2 ||  |2||  |2  ||  |2 ||  |2||  |2
+
| Buffer 4 (10x) ||1,5
|-
|-
-
| Enzyme 1 ||  |0,5 || |0,5 ||  |0,5||  |0,5 || |0,5 ||  |0,5||  |0,5 || |0,5 ||  |0,5||  |0,5 || |0,5 ||  |0,5||  |0,5 || |0,5 ||  |0,5||  |0,5  
+
|Enzyme NotI ||0,5  
|-
|-
-
| Enzyme 2 || |0,5 || |0,5 ||  |0,5||  |0,5 || |0,5 ||  |0,5||  |0,5 || |0,5 ||  |0,5||  |0,5 || |0,5 ||  |0,5||  |0,5 || |0,5 ||  |0,5||  |0,5
+
|H2O || 1,5  
-
|-
+
-
|H<sub>2</sub>O||  | 11||  | 9||  |8||  | 9||  | 6||  |8||  | 9||  | 9||  |9||  | 9||  | 9||  |9||  | 9||  | 11,5||  |11,5||  | 11
+
|-
|-
 +
|'''Total volume /µl'''||15
 +
|}
 +
<br />
 +
{| border="1"
 +
|align="left" | '''Sample-No.''' || align="left" | '''1+2''' ||align="left" | '''3+4''' ||align="left" | '''5+6''' ||align="left" | '''7+8''' ||align="left" | '''9+10''' ||align="left" | '''11+12''' ||align="left" | '''13+14''' ||align="left" | '''15+16''' ||align="left" | '''17+18''' ||align="left" | '''19+20''' ||align="left" | '''21-23''' ||align="left" | '''24+25''' ||align="left" | '''26+27''' ||align="left" | '''28+29''' ||align="left" | '''30''' ||align="left" | '''31'''
 +
|-
 +
|align="left" | '''Components''' ||align="left"| '''453 BAP''' ||align="left"| '''587 BAP''' ||align="left"| '''587 KO BAP'''||align="left"| '''453 RGD'''||align="left"| '''587 RGD'''||align="left"| '''587 KO RGD'''||align="left"| '''453 HIS'''||align="left"| '''587 HIS'''||align="left"| '''587 KO HIS'''||align="left"| '''587 KO EMPTY'''||align="left"| '''453 Z34C'''||align="left"| '''587 Z34C'''||align="left"| '''587 KO Z34C'''||align="left"| '''587 KO Z34C SPACER'''||align="left"| '''pSb1C3_Bla (cut version)'''||align="left"| '''pSb1C3_Bla (uncut)'''
 +
|-
 +
| align="left" | Oligo 1||align="left"|O124: 10 ||align="left"|O126: 10 ||align="left"|O128: 10 ||align="left"|O130: 10 ||align="left"|O132: 10 ||align="left"|O134: 10 ||align="left"|O135: 10 ||align="left"|O137: 10 ||align="left"|O139: 10 ||align="left"|O141: 10 ||align="left"|O143: 10 ||align="left"|O145: 10 ||align="left"|O147: 10 ||align="left"|O149: 10 ||align="left"|--- ||align="left"|---
 +
|-
 +
| align="left" | Oligo 2 ||align="left"|O125: 10 ||align="left"|O127: 10 ||align="left"|O129: 10 ||align="left"|O131: 10 ||align="left"|O133: 10 ||align="left"|O151: 10 ||align="left"|O136: 10 ||align="left"|O138: 10 ||align="left"|O140: 10 ||align="left"|O142: 10 ||align="left"|O144: 10 ||align="left"|O146: 10 ||align="left"|O148: 10 ||align="left"|O150: 10 ||align="left"|--- ||align="left"|---
 +
|-
|}
|}
-
<br />
+
<br/>
 +
Incubation time: 1 h, Incubation temperature: 37°
 +
<br/>
 +
<b>Preparation of gel:</b><br/>
 +
1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 45 minutes
<br />
<br />
-
[[File:Freiburg10 Rep40-78 mVenus.jpg|700px]]
+
<br/>
 +
[[File:Freiburg10_LoopInsertions.jpg|600px]]
<br />
<br />
 +
 +
<br/>
 +
<b>Expected fragment sizes /bp:</b> <br/>
 +
pSB1C3: 2051 bp
 +
Bla: 905<br/>
 +
BAP: ~150<br/>
 +
RGD: ~ 130<br/>
 +
Z34C: ~ 200<br/>
 +
His: ~ 125 <br/>
 +
<br/>
 +
 +
The following clones were sent for sequencing: 2,3,5,7,9,11,13,16,17,19 and 25.
<br />
<br />
-
<br />
+
<p style="font-size:13px; color:#003399;"><b>Comment</b>: Clones (see above) were sent for sequencing with VR2 Primer. In addition, 8 mL DYT medium was inoculated with each of the clones for preparation of glycerol stocks. For sequencing results go to labday 01.09.10.</p>
-
comment: Rep- and AAP approaches have to be repeated. We did not realize that the vector can religate! Plasmids and glycerol stocks were thrown away.
+
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Inoculation of CD colony</b></p>====
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Midi-Prep of pAAV_RC & pHelper</b></p>====
-
Investigator: Kira
+
-
One colony was picked from agar plate and dispersed into 10 ml DYT+ Amp and incubated over night @ 37 C.
+
'''Investigators: Chris W. <br>
 +
<p style="font-size:13px; color:#003399;"><b>Comment</b>: Midi-Preps of pHelper=P356 and pAAV_RC=P357</p>
 +
The Midi-Preps were performed according to the standard protocol yielding the following concentrations:
 +
{| border="1"
 +
| plasmid-no. || align="right" |P356|| align="right" |P357
 +
|-
 +
| concentration (ng/µl)|| align="right" |1068,0 || align="right" |1274.80
 +
|-
 +
|}
 +
<br>
 +
====<p style="font-size:15px; background-color:#66bbff;"><b>Transformation of Biobrick CD</b></p>====
 +
<b>Investigator: Kira</b>
 +
<br/>
 +
Transformation was performed according to the standard protocol.
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<html>
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</div>
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Revision as of 11:52, 6 September 2010

Contents

99. labday 24.08.2010

Sequence analysis of pSB1C3_CFP_middlelinker

Investigator: Jessica


Freiburg10 pSB1C3 CFP middlelinker.jpg

  • pSB1C3_CFP_middlelinker is ready


Harvest viral particles, Transduction of HT1080

Investigator: Kerstin

  • Harvest viral particles following the standard protocol
  • Transduction of three 6-well plates:

Plate 1 YFP: 150.000 cells per well

1 2 3
A control, no cells 500µl virus (1) 500µl virus (2)
B control, no virus 500µl virus (1) 500µl virus (2)

Plate 2 YFP: 150.000 cells per well

1 2 3
A control, no cells 500µl virus (3) 500µl virus (4)
B control, no virus 500µl virus (3) 500µl virus (4)

Plate 3 YFP: 200.000 cells per well

1 2 3
A control, no cells 500µl virus (5) 500µl virus (9)
B control, no virus 500µl virus (9) 500µl virus (5)


  • (1)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,06
  • (2)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,08
  • (3)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,10
  • (4)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,12
  • (5)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,14
  • (9)= 10µg RC, 10µg pHelper, 3,3µg GOI (eGFP)

Mini-prep of SDM NgoMIV CD

Investigator: Kira
Motivation: Harvest DNA for further analysis, e.g. test digestion

Mini-prep was performed on 3 colonies.

c(clone1) = 358, 05 ng/ul c(clone2) = 352, 10 ng/ul c(clone 3) = 376, 53 ng/ul

Sent for sequencing: pSB1C3_6xHis

Investigator: Jessica

  • Plasmid: P84 c= 107,8 ng/µl
  • Primer: VR2
  • tube number: JG84

Subcloning Cap into pAAV_RC_ins-rep

Investigator: Stefan

Comment: Sequencing of pAAV_RC_final showed that Cap was not inserted succsessfully. Therefore it has to be repeated.

Digestion of vector and insert:

  • Insert: pMA_RepCap Vector_SDM_InsPvuII clone 1 (P211)
  • Vector: pAAV_RC_ins-rep clone1 (P250)



A two-step digestion was performed:

1st step

P211 / µl P250 / µl
DNA5,8 5,2
buffer 32 2
Enzyme BsiWI1 1
H2O11,2 11,8
total volume20 20


2nd step

P211 / µl P250 / µl
Mix obtained from step 1 20 20
Enzyme BspMI1 1
H2O1,25 1,25
total volume22,25 22,25


Samples were loaded on a 0,8% agarose gel an run at 115V for about 60 minutes.


pAAV RC final
Freiburg10 Cap Insertion.png


Gelextraction:

Gelex was performed according to protocol exept for elution was done with 60 µl instead of 20 µl.

  • c(P211)= 1,14 ng/µl
  • c(P250)= 5,60 ng/µl



T4 Ligation:
T4 ligation was performed according to protocol. used DNA amounts:

  • v(P211)= 2,56 µl
  • v(P250)= 5,44 µl



Transformation:

Trafo was performed according to protocol using XL1b cells.

Test digestion of pCerulean (p273)

Investigator: Anissa

Comments:Sequencing of pCerulean showed strange results, because the qualtity of sequenzing was not good. For working further with pCerulean, we started with a test digestion, to be sure, everything works.
Because the sequencing showed no AgeI, we cut one time with AgeI and EcoRI, the other time with NotI.


Components p273/µL p273/µL
DNA 2 2
BSA (10x) 1,51,5
Buffer 4 (10x) 1,5 1,5
Enzyme 1 0,5 NotI0,5 EcoRI
Enzyme 2 0,5 NotI 0,5 AgeI
H2O 9 9
Total volume 1515


test digestion of pCerulean

Comments: Results of digestion seem to be all right

Cloning of VP1up into PCerulean

Investigator: Anissa
VP1up was cut out of pSB1C3 and into pCerulean

  • Digestion:

    Components Vector/µL Insert/µL
    DNA 3,78,4
    BSA (10x) 1,52
    Buffer 4 (10x) 1,5 2
    Enzyme 1 EcoRI 1 1
    Enzyme 2 PstI 1 1
    H2O 6,3 5,6
    Total volume 15 20


  • Gel:
    A 1% agarose-gel was made, after 30 minutes samples were cut
    test digestion of pCerulean
  • Gelextraction was performed according the standardprotocol
  • Ligation:
    Components used volume for T4 ligation/µL concentration /ng/µl
    Vector 5,1527,7
    Insert 2,8515,4

    In addition 1µl T4-ligase and 1µl T4-ligase-buffer were added

  • Transformation: was performes into Xl1 blue according the standard-protocol


Sequence analysis of sequencing reads prepared yesterday (23.08.2010)

Investigator: Bea

GENERAL COMMENT: pSB1C3 {NLS and VP1up} were sent for sequencing. Aswell as the pAAV_RC_final which contains all four mutations in the Rep-Cap gene and the integrated and synthesiezd rep and cap gene and the plasmid pCerulean whith the CMV promoter and sv40 polyadenylation site.


Comments: Sequence analysis of pAAV_RC_final containing the subcloned "rep" (ordered) and "Cap (ordered) plus the four mutations to delete the restriction sites PstI, BamHi and SalI.

  • Primer used:
    • VP1 primer for pKex
    • GATC_std_SK
    • Primer Cap 2800 rev
    • Primer Cap 2800 for
    • Primer Cap 3500 for
  • Plasmid sequenced: P283
  • Sequence sample: iGEM4
  • Stored in Geneious-folder: RepCap insert ordered
pAAV RC final
Freiburg10 Seqanalysis pAAV RC final 2010 23 08.jpg


Results:

Sequencing of the mutation ok. Rep integration worked. Cap integration needs to be repeated.


Next steps: Repetition of the integration of the synthesized cap gene performed by Stefan (see topic).

Comments: Sequence analysis of pSB1C3_NLS

  • Primer used: VR-2
  • Plasmid sequenced: P282
  • Sequence sample: iGEM3_O51_VR-2
  • Stored in folder: pSB1C3_NLS
pSB1C3_NLS
Freiburg10 Sequence analysis pSB1C3 NLS 2010 23 08.jpg


Results:

Insertion of the nuclear localisation signal (NLS) into the pSB1C3_NLS as one step in the N-terminal insertion of targeting molecules. Sequencing results look good. prefix and suffix ok!


Next steps: Fuse NLS to targeting molecule.

Comments: Sequence analysis of pSB1C3_VP1up

  • Primer used: VF-2
  • Plasmid sequenced: P280
  • Sequence sample: iGEM1_VF-2
  • Stored in folder: N-Terminal targeting --> pSB1C3_VP1up
pSB1C3_VP1up
Freiburg10 Seqanalysis pSB1C3 VP1up 2010 23 08.jpg


Results:

Prefix ok, but annotation is wrong. VP1up should start with: GC!! Suffix ok! The additional EcoRI before the prefix standard can be due to the bad sequence read quality.


Next steps: Clone VP1up into pCerulean in order to obtain the plasmid with a CMV promoter and the sv40 polyadenylation site.

Comments: Sequence analysis of pCerulean

  • Primer used: GATC_CMV-F
  • Plasmid sequenced: P273
  • Sequence sample: iGEM2_CMV-F
  • Stored in folder: N-Terminal targeting --> pCerulean
pCerulean
Freiburg10 Seqanalysis pCerulean 2010 23 08.jpg


Results:

It seem that the suffix is not correct which means that AgeI, PstI and NotI is missing. This can be due to the bad sequence quality. Prefix is ok!


Next steps: For verification a test digestion with the missing restriction sites will be perforemd and another round of sequencing will be conducted.

[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]

Test digestion of SDM NgoMIV CD

Investigator: Kira
Motivation: In order to check if site directed mutagenesis was successfull, test digestion was performed with 3 clones, which were picked yesterday as well as with 'original' DNA, which still contains all the restriction sites

  • Test digestion:

    Components clone 1/µL clone 2/µL clone 3/µL original/µL
    DNA (500ng) 1,41,4 1,4 1,4
    BSA (10x) 1 1 1 1
    Buffer 4 (10x) 1 1 1 1
    Enzyme 1 NgoMIV 0,5 0,50,50,5
    H2O 6,1 6,1 6,1 6,1
    Total volume 10 10 10 10



    Bitte schneid mich aus !

    Freiburg10 test digestion NgoMIV.jpg

    According to the gel, NgoMIV SDM was successful in clones 1 and 2. Clone 3 well reveales 2 bands of unknown origin. Samples 1 and 3 were sent for sequencing.

    Picking clones of pSB1C3_Affibody_linker and pSB1C3_ß-Globin_YFP_hGH_rITR

    Investigator: Anna

    Comments: A 1:1000 dilution of pSB1C3_Affibody_GSAT-Linker was prepared.
    To do: Mini-Prep of pSB1C3_Affibody_(Short/Middle/Long and SEG-Linker) and pSB1C3_ß-Globin_YFP_hGH_rITR. Picking clones of pSB1C3_Affibody_GSAT-Linker.

    Test digestion of pSB1C3_SDM_SspI_Bla14FM (P222), pSB1C3_BLA (P286, P288)

    Investigator: Achim

    CommentsNew test digestion of two SDM-attempts, this time we also digested the original vector containing the PvuII restriction site to see differences.



    Freiburg10 Test digestion of pSB1C3 BLA final 24 08 2010.jpg


    • The original vector is being cut as expected: the uncut vector can be seen in different conformations, the linearized vector shows one distinct band and the digestions cutting out BLA show a band at ~900 bp. Our SDM attempts show inconsistent results, not only does the Pvu site still seem to be in the backbone, there are no more proper insert bands visible either. We therefore conclude that either the DpnI digestion or the dilution of the transformation must have gone wrong (differing plasmids...). Tomorow we'll try a new cloning approach using an old vector with deleted Pvu restriction site and MscI & XbaI digestion. In case this also fails we also yet have to prep and test digest the repetition of the mutagenesis which was carried out by Stefan.


    </ul>

    pSB1C3_VP1up
    Freiburg10 Seqanalysis pSB1C3 VP1up 2010 23 08.jpg


    Results:

    Prefix ok, but annotation is wrong. VP1up should start with: GC!! Suffix ok! The additional EcoRI before the prefix standard can be due to the bad sequence read quality.


    Next steps: Clone VP1up into pCerulean in order to obtain the plasmid with a CMV promoter and the sv40 polyadenylation site.

    Comments: Sequence analysis of pCerulean

    • Primer used: GATC_CMV-F
    • Plasmid sequenced: P273
    • Sequence sample: iGEM2_CMV-F
    • Stored in folder: N-Terminal targeting --> pCerulean
    pCerulean
    Freiburg10 Seqanalysis pCerulean 2010 23 08.jpg


    Results:

    It seem that the suffix is not correct which means that AgeI, PstI and NotI is missing. This can be due to the bad sequence quality. Prefix is ok!


    Next steps: For verification a test digestion with the missing restriction sites will be perforemd and another round of sequencing will be conducted.

    [http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]

    Test digestion of SDM NgoMIV CD

    Investigator: Kira
    Motivation: In order to check if site directed mutagenesis was successfull, test digestion was performed with 3 clones, which were picked yesterday as well as with 'original' DNA, which still contains all the restriction sites

  • Test digestion:

    Components clone 1/µL clone 2/µL clone 3/µL original/µL
    DNA (500ng) 1,41,4 1,4 1,4
    BSA (10x) 1 1 1 1
    Buffer 4 (10x) 1 1 1 1
    Enzyme 1 NgoMIV 0,5 0,50,50,5
    H2O 6,1 6,1 6,1 6,1
    Total volume 10 10 10 10



    Bitte schneid mich aus !

    Freiburg10 test digestion NgoMIV.jpg

    According to the gel, NgoMIV SDM was successful in clones 1 and 2. Clone 3 well reveales 2 bands of unknown origin. Samples 1 and 3 were sent for sequencing.

    Picking clones of pSB1C3_Affibody_linker and pSB1C3_ß-Globin_YFP_hGH_rITR

    Investigator: Anna

    Comments: A 1:1000 dilution of pSB1C3_Affibody_GSAT-Linker was prepared.
    To do: Mini-Prep of pSB1C3_Affibody_(Short/Middle/Long and SEG-Linker) and pSB1C3_ß-Globin_YFP_hGH_rITR. Picking clones of pSB1C3_Affibody_GSAT-Linker.

    Test digestion of pSB1C3_SDM_SspI_Bla14FM (P222), pSB1C3_BLA (P286, P288)

    Investigator: Achim

    CommentsNew test digestion of two SDM-attempts, this time we also digested the original vector containing the PvuII restriction site to see differences.



    Freiburg10 Test digestion of pSB1C3 BLA final 24 08 2010.jpg


    • The original vector is being cut as expected: the uncut vector can be seen in different conformations, the linearized vector shows one distinct band and the digestions cutting out BLA show a band at ~900 bp. Our SDM attempts show inconsistent results, not only does the Pvu site still seem to be in the backbone, there are no more proper insert bands visible either. We therefore conclude that either the DpnI digestion or the dilution of the transformation must have gone wrong (differing plasmids...). Tomorow we'll try a new cloning approach using an old vector with deleted Pvu restriction site and MscI & XbaI digestion. In case this also fails we also yet have to prep and test digest the repetition of the mutagenesis which was carried out by Stefan.


    100. labday 25.08.2010

    Cloning of pSB1C3_Rep40, pSB1C3_Rep68 and pMA_RC_insert

    Investigator: Chris L.

    • buffer used: 2; Restriction-enzymes used: Enzyme 1: HindIII ; Enzyme 2: BstEII

    Plasmids:

    • pSB1C3_Rep40 P231
    • pSB1C3_Rep68 P266

      • Insert:

    • pMA_RC_insert P190


    Components P190/µL P231/µL P266/µL
    DNA 5.9 6.4 5
    BSA (10x) 2 2 2
    Buffer 2 (10x) 2 2 2
    Enzyme 1 HindIII 1 1 1
    Enzyme 2 BstEII 1 1 1
    H2O 8.1 7.6 9
    Total volume 20 20 20


    • Incubation: 90 minutes; 37°C with HindIII
    • Incubation: 90 minutes; 60°C with BstEII


    Agarosegel
    0.5 g Agarose, 50 ml TAE (1 %), 3 µL GELRED, 5 min at 90 Volt, 40 min at 115 Volt
    Freiburg 10 pSB1C3 Rep40 pSB1C3 Rep68 pMA RC Insert.png
    Concentrations measured via NanoDrop:

    • pSB1C3_Rep40: 81.97 ng/µl
    • pSB1C3_Rep68: 48,68 ng/µl
    • RC_Insert: 3,60 ng/µl


    T4 Ligation of pSB1C3_Rep40 with RC_Insert
    Volume insert: 6,54 µl
    Volume vector: 1,46 µl

    T4 Ligation of pSB1C3_Rep68 with RC_Insert
    Volume insert: 5,46 µl
    Volume vector: 2,54 µl

    Trafo was prepared with XL1blue and Cm

    Picking clones of pAAV_RC-ins_rep_cap and pCeruleanVP1up

    Investigator: Chris L.

    To do: Mini-Prep of pAAV_RC-ins_rep_cap and pCeruleanVP1up.

    Cloning of pSB1C3_6xHis with middlelinker and pSB1C3_6xHis with pGA14_mVenus_YFP

    Investigator: Jessica

    • buffer used: 4; Restriction-enzymes used: Enzyme 1: AgeI ; Enzyme 2: PstI ; Enzyme3: NgoMIV
    • Plasmids:
      • pSB1C3_6xHis P84
      • pGA14_middle linker P65
      • GA14_mVenus_YFP P60



    Components Mastermix P84/µL P65/µL P60/µL
    DNA - 13,91 13,52 8,93
    BSA (10x) -2 2 2
    Buffer 4 (10x) - 2 2 2
    Enzyme 1 AgeI - 1 - -
    Enzyme 2 PstI - 1 1 1
    Enzyme 3 NgoMIV - - 1 1
    H2O - 0,09 0,48 5,07
    Total volume - 20 20 20


    • Incubation:110 minutes; 37°C

    Agarosegel
    0.5 g Agarose, 50 ml TAE (1 %), 3 µL GELRED, at 120 Volt



    Freiburg10 gel cut n terminus1.jpg


























    concentrations measured via NanoDrop:

    • SB1C3_6xHis: 1,7 ng/µl
    • middle linker: 3,8 ng/µl
    • mVenus_YFP: 3,2 ng/µl


    T4 Ligation pSB1C3_6xHis_middlelinker
    Volume insert: 0,25 µl
    Volume vector: 7,75 µl

    T4 Ligation pSB1C3_6xHis_mVenus_YFP
    Volume insert: 2,64 µl
    Volume vector: 5,36 µl

    Trafo was prepared with XL1blue and Cm

    Sequence analysis of pSB1C3_6xHis

    Investigator: Jessica
    P84 was sequenced for continuation of working with it


    Freiburg10 pSB1C3 6xHis.jpg

    • pSB1C3_6xHis P84 is ready to use, but is empty. a new tube will be made tomorrow

    Inoculation of pGA14_middlelinker and pSB1C3_6xHis clone1

    Investigator: Jessica

    • both tubes (P65 and P84)are empty --> inoculation with glycerolstocks
      • B48 pGA14_middlelinker was inoculated with 10ml DYT and 10µl Amp
      • B64 pSB1C3_6xHis clone1 was inoculated with 10ml DYT and 10µl Cm

    Cloning of pSB1C3_SspI_BLA and pSB1C3_CFP_PvuII

    Investigator: Achim

    Comment:I ligated a sequence from pSB1C3_CFP_PvuII missing the PvuII restriction site into pSB1C3_SspI_BLA to obtain a pSB1C3 vector without PvuII in the cat marker.

    Vector:

    • pSB1C3_SspI_BLA (p222): c= 308,32 ng/µl

    Insert:

    • pSB1C3_CFP_PvuII (p129): c= 264,86 ng/µl


    components Vector Insert
    DNA 3,8 4,9
    BSA (10x) 2 2
    Buffer 4 (10x)2 2
    Enzyme MscI 1 1
    Enzyme XbaI 1 1
    H2O10,2 9,1
    Total volume 20 20


    • Gelextraction:

    0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 110 Volt, running time:45
    Marker: GeneRuler ladder mix (Fermentas)



    Freiburg10 25082010achim.JPG


    • c(Insert)= 15,66 ng/µl; size: 730 bp
    • c(Vector)= 14,42 ng/µl; size: 2200 bp


    • Ligation of PCR products and vector:

    For the Ligation 1µl T4 buffer (2x) and 1µl T4 ligase were used. Incubation time: 60 min due to blunt end ligation

    vector /µl insert /µl
    pSB1C3_BLA 0,86 7,14


    • Transformation:

    The transformation was done following the standard protocol using XL1 blue cells.

    Comment: Two clones were picked, but because we already obtained our plasmid via SDM in the meantime, no prep was performed. Glycerol stocks of the two clones were created just in case, B264 and B265.

    Mini-Preps and test digestion of pSB1C3_Affibody_Middle-Linker, pSB1C3_Affibody_Short-Linker, pSB1C3_Affibody_SEG-Linker, pSB1C3_Affibody_Long-Linker and pSB1C3_betaglobin_mVenus_hGH_rITR

    Investigator: Stefan

      Glycerol stocks were prepared:
    • B240 = pSB1C3_Affibody_Middle-Linker clone1
    • B241 = pSB1C3_Affibody_Middle-Linker clone2


    • B242 = pSB1C3_Affibody_Short-Linker clone 1
    • B243 = pSB1C3_Affibody_Short-Linker clone 2


    • B246 = pSB1C3_Affibody_SEG-Linker clone1
    • B247 = pSB1C3_Affibody_SEG-Linker clone2


    • B248 = pSB1C3_Affibody_Long-Linker clone 1
    • B249 = pSB1C3_Affibody_Long-Linker clone 2


    • B244 = pSB1C3_betaglobin_mVenus_hGH_rITR clone 1
    • B245 = pSB1C3_betaglobin_mVenus_hGH_rITR clone 2



      • Mini-Prep following the standard protocol

    • P290 = pSB1C3_Affibody_Middle-Linker clone1 c= 227,36 ng/µl
    • P291 = pSB1C3_Affibody_Middle-Linker clone2 c= 219,05 ng/µl


    • P292 = pSB1C3_Affibody_Short-Linker clone 1 c= 196,63 ng/µl
    • P293 = pSB1C3_Affibody_Short-Linker clone 2 c= 224,76 ng/µl


    • P296 = pSB1C3_Affibody_SEG-Linker clone1 c= 218,68 ng/µl
    • P297 = pSB1C3_Affibody_SEG-Linker clone2 c= 229,66 ng/µl


    • P298 = pSB1C3_Affibody_Long-Linker clone 1 c= 229,73 ng/µl
    • P299 = pSB1C3_Affibody_Long-Linker clone 2 c= 202,05 ng/µl


    • P294 = pSB1C3_betaglobin_mVenus_hGH_rITR clone 1 c= 425,14 ng/µl
    • P295 = pSB1C3_betaglobin_mVenus_hGH_rITR clone 2 c= 328,28 ng/µl


    Test digestion

    • Restriction-enzymes used: for P290-P293 and P296-P299: NotI ; for P294-P295: NgoMIV and PstI

    comment: The same amount of ingredients were used for P290-P293 and P296-P299, therefore these approaches will be merged into the chart. The same goes for P294-P295.

    Components P290-P293 and P296-P299 P294-P295
    DNA 1 1
    BSA (10x) 1 1
    Buffer 4 (10x) 1 1
    NotI 0,5 -
    NgoMIV - 0,5
    PstI - 0,5
    H2O 6,5 6
    Total volume 10 10


    Incubation time: 70 minutes; Incubation temperature: 37°
    0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , running time:5 minutes at 90 Volt and 50 minutes at 115 Volt
    2µl loading dye (6x) for the sample, Marker: GeneRuler ladder mix (Fermentas)

    Freiburg10 test digestion affi+linker right assembly.jpg

    Comment: Sizes of fragments look like expected. Clone 1 of each plasmid will be sent for sequencing tomorrow.

    Sequenzing evaluation of SDM NgoMIV

    Investigator: Kira

    2 samples have been sent for sequencing yesterday. According to the data, both samples do not contain any NgoMIV restriction site anymore.


    Freiburg10 sample 1, seq 1.jpg


    Freiburg10 sample 3, seq2.jpg

    101. labday 26.08.2010

    Mini-Preps of pGA14_middle linker and pSB1C3_6xHis clone 1

    Investigator: Jessica

      Glycerol stocks were not prepared because I have inoculated from glycerol stocks
      Mini-Prep following the standard protocol
    • P301 = pGA14_middle linker (= P65)
      c=305,7


    • P300 = pSB1C3_6xHis clone 1 (= P84)
      c=177,5



    Mini-Preps and test digestion of pAAV_RC-ins_rep_cap, pCeruleanVP1up and pAAV_RC_ins-rep-cap.

    Investigator: Chris L.

      Glycerol stocks were prepared:
    • B250 = pCerulean_VP1up clone1
    • B251 = pCerulean_VP1up clone2
    • B252 = pCerulean_VP1up clone3
    • B253 = pCerulean_VP1up clone4
    • B254 = pSB1C3_VCK_Bla clone1
    • B255 = pSB1C3_VCK_Bla clone2
    • B256 = pSB1C3_VCK_Bla clone3
    • B257 = pSB1C3_VCK_Bla clone4
    • B262 = pAAV_RC_ins-rep-cap clone1
    • B263 = pAAV_RC_ins-rep-cap clone2


      • Mini-Prep following the standard protocol

    • P302 = pCerulean_VP1up clone1
      c=410,70 ng/µl
    • P303 = pCerulean_VP1up clone2
      c=387,78 ng/µl
    • P304 = pCerulean_VP1up clone3
      c=321,04 ng/µl
    • P305 = pCerulean_VP1up clone4
      c=350,34 ng/µl
    • P306 = pSB1C3_VCK_Bla clone1
      c=82,53 ng/µl
    • P307 = pSB1C3_VCK_Bla clone2
      c=92,70 ng/µl
    • P308 = pSB1C3_VCK_Bla clone3
      c=82,22 ng/µl
    • P309 = pSB1C3_VCK_Bla clone4
      c=105,34 ng/µl
    • P314 = pAAV_RC_ins-rep-cap clone1
      c=517,04 ng/µl
    • P315 = pAAV_RC_ins-rep-cap clone2
      c=444,33 ng/µl


    Test digestion:

    components volume of P302/µl volume of P303/µlvolume of P304/µl volume of P305/µl volume of P306/µlvolume of P306/µlvolume of P306µl volume of P307/µlvolume of P307µl volume of P307/µlvolume of P308/µlvolume of P308/µl volume of P308/µl volume of P309/µl volume of P309/µl volume of P309/µlvolume of P314/µl volume of P315/µl
    DNA 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 1 1
    BSA (10x) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
    Buffer 4 (10x) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 - -
    Buffer 2 (10x) - - - - - - - - - - - - - - -- 1 1
    Enzyme NotI - - - - 0,5 - - 0,5 - - 0,5 - - 0,5 - - - -
    Enzyme SspI - - - - - 0,5 - - 0,5 - - 0,5 - - 0,5 - - -
    Enzyme SalI - - - - - - 0,5 - - 0,5 -- 0,5 - - 0,5 - -
    Enzyme BamHI - - - - - - 0,5 - - - - - - - - - --
    Enzyme PvuII - - - - - - 0,5 - - - - - - - - - --
    Enzyme Acc65I - - - - - - - - - - - - - - -- 0,75 0,75
    Enzyme XcmI - - - - - - - - - - - - - - -- 0,5 0,5
    Enzyme PstI 0,5 0,5 0,5 0,5 - - - - - - - - - - - - - -
    Enzyme EcoRI 0,5 0,5 0,5 0,5 - - - - - - - - - - - - - -
    H2O 6 6 6 6 5,5 5 5 5,5 5 5 5,5 55 5,5 5 5 5,75 5,75
    Total volume /µl10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10


    Incubation time: 1 h, Incubation temperature: 37°
    Preparation of gel:
    1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 50 minutes

    Freiburg10 test digestion pSB1C3 VCK Bla+pCerulean Vp1up.jpg

    Results:

    pSB1C3_VCK_Bla: Test digestion looks like expected. Clone 4 was sent for sequencing.
    pCerulean_VP1up: Gel looks well as well. clone 2 was sent for sequencing.
    pAAV_RC_ins-rep-cap: Test digestion looks strange. Just one big band with more than 10000 bp and one very small with 150 bp. Maybe the restriction enzymes didn´t cut. (@Christian: please insert picture)

    102. labday 27.08.2010

    Sequence analysis of pSB1C3_Bla_final (P309)and pCerulean_VP1up (P303)

    Investigator: Bea

    GENERAL COMMENT: pSB1C3_Bba_final (P309, SB_9) and pCerulean_Vp1up (P303; SB_10) were sent for sequencing. In the case of SB1C3_Bba_final, which means that the whole vector was assembled from the buttom,the purpose was to test the designed primers. In the other case we wanted to verify the insertion of Vp1up inot pCerulean.


    Comments: Sequence analysis of pSB1C3_leftITR_CMV_betaglobin_mVenus_hGH_rightITR.

    • Primer used:
      • pSB1C3_Bba_seq_for
      • pSB1C3_Bba_seq_rev
    • Plasmid sequenced: P??
    • Sequence sample: ??
    • Stored in Geneious-folder: BioBricks --> final parts
    pSB1C3_leftITR_CMV_betaglobin_mVenus_hGH_rightITR
    Freiburg10 Seqanalysis pSB1C3 Bba final 2010 27 08.jpg


    Results:

    Results look good. The forward primer worked, still waiting for the results of the reverse primer. The scar inbetween the CMV promoter and the beat globin intron corresponds to the expected result.


    Next steps: Wwait until sequencing results of the reverse primer can be confirmed aswell. If that will not be the case, order new sequensing primer.

    [http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]

    Comments: Sequence analysis of pCerulean_Vp1up.

    • Primer used: CMV-F
    • Plasmid sequenced: P303
    • Sequence sample: ??
    • Stored in Geneious-folder: N-terminal Targeting --> pCerulean_Vp1up
    pCerulean_Vp1up
    Freiburg10 Seqanalysis pCerulean Vp1up 2010 27 08.jpg


    Results:

    Results are good. Insertion of the Vp1up region can be confirmed.


    Next steps: Fuse pCerulean_Vp1up to the NLS.

    [http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]

    Hybridisation and cloning of loop insertions

    Investigator: Achim, Volker

    We hybridized the oligos for the different loop insertions and cloned them into the pSB1C3_BLA backbone.

    Hybridisation

    • 4 Inserts contained overlapping ends and had to be filled up using Klenov fragments. We therefore added Klenow buffer and dNTPs to those samples.
    Components 453 BAP 587 BAP 587 KO BAP 453 RGD 587 RGD 587 KO RGD 453 HIS 587 HIS 587 KO HIS 587 KO EMPTY 453 Z34C 587 Z34C 587 KO Z34C 587 KO Z34C SPACER
    Oligo 1O124: 10 O126: 10 O128: 10 O130: 10 O132: 10 O134: 10 O135: 10 O137: 10 O139: 10 O141: 10 O143: 10 O145: 10 O147: 10 O149: 10
    Oligo 2 O125: 10 O127: 10 O129: 10 O131: 10 O133: 10 O151: 10 O136: 10 O138: 10 O140: 10 O142: 10 O144: 10 O146: 10 O148: 10 O150: 10
    TrisHCl pH8444444 4444----
    5mM MgCl2888888 8888----
    Klenow Buffer------ ----4444
    dNTP Mix------ ----1111
    H2O888888 888815151515
    Total volume 40 40 40 40 40 40 40 40 40 40 40 40 40 40
    • Hybridisation was carried out according to standard protocoll
    • Klenov fill-in reaction:
      • Added 1 µl of NEB Klenow fragment to samples 11,12,13,14
      • incubated for 1h @ 37°C

    Digestion of pSB1C3_BLA vector and Samples 11-14

    • We digested the standard vector with the 453 (Ssp/Sal) and the 587 (Bam/Pvu) standard. The samples that were filled in were also digested to create sticky ends.
    Components V453 V587 11 12 13 14
    DNA 2,5 2,5 3,5 3,5 3,5 3,5
    BSA (10x) - - - - - -
    Buffer 4 (10x) 2 2 2 2 2 2
    Enzyme 1 Ssp: 1 Bam: 1 Ssp: 1 Bam: 1 Bam: 1 Bam: 1
    Enzyme 2 Sal:1 Pvu: 1 Sal: 1 Pvu: 1 Pvu: 1 Pvu: 1
    H2O 13,5 13,5 12,5 12,5 12,5 12,5
    Total volume 20 20 20 20 20 20

    Gel Extraction

    Freiburg10 27082010achim.JPG

    Ligation

    Cloning of pCerulean_VP1up_NLS

    Investigator: Anissa

    Comment:Cloning did not work, because no NLS-band could be seen in the gel... Will be repeated on monday in two new approaches. One time VP1up will be cloned into pSB1C3_NLS and recloned as a fusion-product into pCerulean. Another time the oligos of NLS will be hybridisized, cut with rescriction enzymes and purificated with the Qiaex II kit.Then the NLS will be ligated into the pCerulean_VP1up


    • Digestion:
    components Vector Insert
    DNA 2,6 32
    BSA (10x) 1,5 -
    Buffer 4 (10x)1,5 4
    Enzyme 1 PstI 2 PstI
    Enzyme 1 AgeI 2 NgoMIV
    H2O7,4 -
    Total volume 15 40


    • Gel: that's only the picture of the 2% agarose gel for seperating the NLS. But no band could be seen.

    Freiburg10 NLS.jpg

    DARPin E_01

    Investigator: Bea

    General overview of DARPins:

    Natural protein ankyrin repeat molecules are motifs which can be found in proteins. These motifs are mediating protein-protein interactions. This suggests that ankyrin repeat (AR) proteins can be used for designing binding molecules. In Kohl et al. (2003) and Binz et al. (2003) the authors designed a structural framework with fixed consensus regions and randomized positions of interacting residues.

    The repetitive nature of the ankyrin proteins allows modifications in their variable and modular binding surface. Therefore consensus sequences of natural ankyrin proteins were used to design novel and stable scaffolds for binding proteins. Designed Ankyrin Proteins (DARPins) are well expressed, monomeric in solution, thermodynamically stable and have the ability to fold fast. In the publication of Steiner et al. (2008) screening libraries were created by useing the signal recognition particle (SRP) translocation pathway for phage display. The proteins containing the appropriate translocation signal sequence are efficiently displayed on filamentous phage particles. Screening for DARPins binding to specific target proteins like EGF-R (Erb1) were performed by phage ELISAs. The extracellular domain I-III of the receptor ErbB1 was fused to the Fc part of human IgG1 and used for selection. The previously described combinatorial N3C library was used as a template for SRP selection. After phage ELISA ans sequencing have been performed only one clone (E_01) could be selected with high-affinity binding characteristics. For broaden the diversity of ErbB1 DARPins three more clones were selected by epitope masking (E_67, E_68 and E_69). Binding and epitope localization experiments showed that all selected clones recognize a competing epitope as the dominant binder E_01, except of E_69 which recognizes an epitope not competing with E_01. The low diversity obtained for DARPins against ErbB1 suggests that the binding interfaces are well suited for dominant selection. Additionally the selected clones showed no cross-reactivity with other ErbB-family receptors (Figure 2C).

    Freiburg10 DARPin E 01 table Steiner et al.jpg


    The dominant DARPpin E_01 has very high affinities to the target protein ErbB1 (Table 3) and can be used as a potential targeting molecule four our approach in fusing the DARPin to the N-terminal VP proteins.

    Overview of DARPinE_01 used in our approach:

    Freiburg10 DARPin E 01.jpg

    The designed ankyrin repeat protein used as a potential targeting molecule consists of three internal capping repeats and the C-and N-terminal capping repeats. Each internal repeat module comprises of one beta-turn and two hydrophobic alpha-helices. The potential interaction residues are located in the beta-turn the first alpha-helices of the AR-proteins. The complete nucleotide and corresponding amino acid sequence can be found in the appendix.

    Freiburg10 DARPin E 01 structure.jpg
    Freiburg10 DARPin E 01 structure 2.jpg


    [http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]

    Update VP1 insertion

    Investigator: Anissa, Bea

    Done:

    • pSB1C3_VP1up was succesfully created by Achim. For details see labday from 2010-22-08.
    • pCerulean_VP1up was suceesfully created by Anissa. pCerulean was sequenced and the VP1up which contains the upstream 137 aa´s of the VP1 protein could be successfully cloned into the plasmid backbone pCerulean (obtained by performeing a PCR).
    • pSB1C3_NLSHybridization of the nuclear localization sequence (NLS) was successfully incorporated into the pSB1C3_CFP.
    • Today, Anissa tried to fuse the NLS to the VP1up sequence. Cutting the pSB1C3_NLS with NgomIV and PstI leads to a 45 nt fragment which normally can be resoluted in a 2% agarose gel. But no fragment could be detected in the gel (for further details see: Cloning of pCerulean_VP1up_NLS).
      On monday another approach will be performed: The NLS oligos will be hybridized and digested with NgomIV and SpeI. With the Qiaex II Kit (protocol for desalting and concentrating DNA solutions),the hybrifized oligos will be purified. Parallel, another digestion approach will be conducted.
      • Overview
      Freiburg10 Overview VP1 part1.jpg
      Freiburg10 Overview VP1 part2.jpg

    Next steps:

    • We obtain pSB1C3_VP23_HSPG_ko by conducting a PCR with the pAAV_RC_final (which contains the integrated "cap".
    • Fuse the obtained construct pSB1C3_Targeting Molcule to the pSB1C3_VP23_HSPG_ko.
      • Affibody ZEGFR:1907
      • DARPin E_01
      • 6xHis Tag
      • CFP
    • Fuse pSB1C3_VP23_HSPG_ko_Targeting molecule to the construct pCerulean_VP1up_NLS.
    • Finally, for providing the possibilty to express obtained construct in trans: perform a site-directed mutagenesis with the pAAV_RC_final to remove the startcodon of VP1.

    [http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]

    Sequence analysis of pSB1C3_BLA_final (P309)

    Investigator: Bea

    GENERAL COMMENT: pSB1C3_BLA_final (P309) was sent for sequencing. the beta lactamse was inserted and two site-directed mutagenesis have been performed in order to delete the loop insertion restriction enzymes in the CAT marker.


    Comments: Sequence analysis of pSB1C3_BLA_final containing the two deleted restriction sites (SspI and PvuII)in the CAT marker.

    • Primer used:
      • pQE-RP
      • VF-2
    • Plasmid sequenced: P309
    • Sequence sample: SB_9
    • Stored in Geneious-folder: pSB1C3_BLA
    pSB1C3_BLA_final
    Freiburg10 Seqanalysis pSB1C3 BLA final P309 SspI.jpg
    Freiburg10 Seqanalysis pSB1C3 BLA final P309 PvuII.jpg
    Freiburg10 Seqanalysis pSB1C3 BLA final P309.jpg


    Results:

    The two performed site-directed mutagenesis performed at the backbone of the pSB1C3 containing the beta-lactamase can be confrimed as it can be seen in the two pictures above. Additionally the BLA sequence was sequenced aswell as the insertion of the BLA which can be confirmed with no mutations.


    Next steps: This new plasmid can be used for further experiments. Clone the PCR prdouct of the pAAV_RC into the pSB1C3 and subclone the loop insertion motifs into the vector.

    [http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]

    Mini-Preps and test digestion of pSB1C3_6xHis_middlelinker and pSB1C3_6xHis_mVenus

    Investigator: Stefan

      Glycerol stocks were prepared:
    • B258 = pSB1C3_6xHis_middlelinker clone1
    • B259 = pSB1C3_6xHis_middlelinker clone2


    • B260 = pSB1C3_6xHis_mVenus clone 1
    • B261 = pSB1C3_6xHis_mVenus clone 2


      • Mini-Prep following the standard protocol

    • P310 = pSB1C3_6xHis_middlelinker clone1
      c= 185,31 ng/µl
    • P311 = pSB1C3_6xHis_middlelinker clone2
      c= 178,87 ng/µl


    • P312 = pSB1C3_6xHis_mVenus clone 1
      c= 247,62 ng/µl
    • P313 = pSB1C3_6xHis_mVenus clone 2
      c= 268,98 ng/µl


    Test digestion

    • Restriction-enzymes used: EcoRI and PstI

    Components P310+311 P312+313
    DNA 2 2
    BSA (10x) 1 1
    Buffer 4 (10x) 1 1
    EcoRI 0,5 0,5
    PstI 0,5 0,5
    H2O 5 5
    Total volume 10 10


    Incubation time: 90 minutes; Incubation temperature: 37°
    1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , running time: 55 minutes at 120 Volt


    Freiburg10 test digestion His middle+His mVenus.jpg

    Comment: Test digestion looks good. Clones 1 of each plasmid (P310 and p312) were sent in for sequencing.

    Repetition of test digestion of pAAV_RC_ins-rep-cap, pAAV_RC_ins-rep, pSB1C3_Rep40_RC_Insert and pSB1C3_Rep68_RC_Insert.

    Investigator: Chris L.

    • P250 = pAAV_RC_ins-rep clone1
      c=467,50 ng/µl
    • P314 = pAAV_RC_ins-rep-cap clone1
      c=517,04 ng/µl
    • P315 = pAAV_RC_ins-rep-cap clone2
      c=444,33 ng/µl


    • P316 = pSB1C3_Rep40_ins clone 1
      c=302,22 ng/µl
    • P317 = pSB1C3_Rep40_ins clone 2
      c=274,37 ng/µl
    • P318 = pSB1C3_Rep68_ins clone 1
      c=491,69 ng/µl
    • P319 = pSB1C3_Rep68_ins clone 2
      c=498,71 ng/µl


    Test digestion:

    components volume of P250/µl volume of P314/µl volume of P315/µl volume of P316/µl volume of P317/µl volume of P318/µl volume of P319/µl
    DNA 1 1 1 1 1 1 1
    BSA (10x) 1 1 1 1 1 1 1
    Buffer 2 (10x) 1 1 1 1 1 1 1
    Enzyme Acc65I 0,75 0,75 0,75 - - - -
    Enzyme XcmI 0,5 0,5 0,5 - - - -
    Enzyme HindIII - - - 0,5 0,5 0,5 0,5
    Enzyme BstEII - - - 0,5 0,5 0,5 0,5
    H2O 5,75 5,75 5,75 6 6 6 6
    Total volume /µl10 10 10 10 10 10 10


    Incubation time: 1 h, Incubation temperature: 37° for P250, P314 and P315
    Incubation time: 45 min, Incubation temperature: 37° with HindIII for P316, P317, P318 and P319 ; 45 min, Incubation temperature: 60° with BstEII for P316, P317, P318 and P319 >br /> Preparation of gel:
    1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 120 Volt, running time: 45 minutes


    • Marker: GeneRuler ladder mix
    Marker Sample P250 10 /µl Sample P314 10 /µl Sample P315 10 /µl Sample P316 10 /µl Sample P317 10 /µl Sample P318 10 /µl Sample P319 10 /µl
    Lane 1 3 4 5 7 8 9 10


    Freiburg10 pAAV RC ins-rep-cap pSB1C3 Rep40 ins pSB1C3 Rep68 ins.png

    Comment: The P314 and P315 looks like the control vector. The RepCap Vector_SDM_InsPvuII insertion didn´t work. The P316, P317, P318 and P318 was cut with the "wrong" enzymes. The same fragment size as the original vector appeared. Next time cut with EcoRI.

    Midi-Prep of pSB1C3_VCK_Bla

    Investigators: Chris W.

    Comment: Midi-Preps of P 320, B257

    The Midi-Preps were performed according to the standard protocol yielding the following concentrations:

    plasmid-no. P320
    concentration (ng/µl)408


    103. labday 28.08.2010

    Mini-Prep of pSB1C3_NLS

    Investigator: Stefan
    comments: No glycerol stock and test digestion was performed because bacteria culture inocculated from glyerol stock (B233).

      Mini-Prep following the standard protocol
    • P321 = pSB1C3_NLS (1)
      c= 180,45 ng/µl
    • P322 = pSB1C3_NLS (2)
      c= 187,26 ng/µl


    COMMENT (Patrick): These mini-preps will be thrown away because B233 was used for inoculation due to a wrong entry in our plasmid excel sheet. B233 this is NOT the sequenced clone ! We sequenced B234/P282. Therefore 2x10 ml DYT + Cm were inoculated with B234.

    Cell culture

    Harvest viral Stock and Transductiot of HT and A431 cells
    1. P169: 100.000 cells
    2. P169: 200.000 cells
    3. P169: 300.000 cells
    4. P169: 400.000 cells
    5. P169: 500.000 cells
    6. P169: 600.000 cells
    7. P169: 700.000 cells
    8. P262: 100.000 cells
    9. P262: 200.000 cells
    10. P262: 300.000 cells
    11. P262: 400.000 cells
    12. P262: 500.000 cells
    13. P262: 600.000 cells
    14. P262: 700.000 cells


    200.000 cells per well

    1 2 3
    A control, no cells 1 2
    B control, no virus 1 2
    1 2 3
    A control, no cells 3 4
    B control, no virus 3 4
    1 2 3
    A control, no cells 5 6
    B control, no virus 5 6
    1 2 3
    A control, no cells 7 8
    B control, no virus 7 9
    1 2 3
    A control, no cells 11 13
    B 10 12 14
    Seeding AAV293 for Transfection at 30.8


    8 plates with 200.000 cells each were seeded

    Picking clones from the ViralBricks

    Investigator: Volker

    Clones from all 14 transformations of the ViralBricks were picked. Because the probability that the ligation worked and that the synthesized oligos do not conatin mutations is quite bad I picked 8 to 10 clones from each ligation, resulting in approximately 150 clones at all. The clones were used to inoculate 5ml of a mixture of 60%LB-media nd 40%DYP-media because our stocks were not sufficient for this experiment.

    104. labday 29.08.2010

    P282/B234 mini-prep

    ... was performed according to the standard protocol.
    Labelling:

    • P323, pSB1C3_NLS clone 2.1, 129 ng/µl
    • P324, pSB1C3_NLS clone 2.2, 141 ng/µl

    Investigator: Patrick

    Digestion of pSB1C3_NLS (P324)

    The NLS was cut out with NgoMIV and EcoRI, therefore the expected size of the hoped-for fragment is 55 bp. About 30 ng DNA can be detected in a 1% gel. According to my calculation there will be maximal 80 ng insert.
    55bp/2019bp = 0,027
    0,027x140 ng/µl = 3,81 ng/µl
    80 ng/3,81 ng/µl = 21 µl

    Digestion: 21 µl pSB1C3_NLS (P324), 2,5 µl EcoRI, 2,5 µl NgoMIV, 3 µl Buffer 4, 1 µl H2O.
    Digestion Time: 2 h 20 minutes.

    The 2% agarose gel was loaded with 36 µl digesteion product including 6 µl 30% glycerol solution. I did not use loading dye because i to prevent that the dye overlay the mutual 55 bp insert.

    Pat2908.JPG

    The gelextraction was performed according to the the standard protocl. The resolution of the picture ist not sufficient to be able to see the band clearly as the gel was put onto the UV-light table a very thin band with the expected size could be seen.
    DNA concentration measurement: 3,28 & 7,7 ng/µl (two measurements). Especially the second value is doubtable because according to my calculation above the insert conccentration cant be higher than 3,81 ng/µl. Labeled: NLS, put into 4°C room.

    New digestion over night following a gelrun tomorrow: 23 µl pSB1C3_NLS (P324), 2 µl EcoRI, 2 µl NgoMIV, 3 µl Buffer 4.

    Investigator: Patrick

    Harvesting cultures from the ViralBricks

    Investigator: Volker

    All approximately 150 cultures were pelletted (twice 2 ml) and the supernatant thrown away. The pellets were freezed and the rest of the culture stored in the 4°C room. These pellets will be preped, test digested and sequenced subsequently until right clones are identified.

    105. labday 30.08.2010

    BioBrick assembly of pSB1C3_leftITR_pTERT & pSB1C3_ß-globin_YFP_hGH_rITR

    Investigator: Achim

    comment: final assembly of a vector containing all aav elements & the tert promoter
    Digestion:

    components: pSB1C3_ß-globin_YFP_hGH_rITR(Vector): P294 pSB1C3_leftITR_pTERT (Insert): P256
    DNA 2,4 8,4
    BSA (10x) 2 2
    Buffer 4 (10x)2 2
    Enzyme1 1 (EcoRI) 1 (EcoRI)
    Enzyme2 1 (XbaI)1 (SpeI)
    H2O11,6 5,6
    Total volume 2020



    Preparation of gel:
    0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 110 Volt, running time: 45 minutes


    Freiburg10 30082010achim2.JPG


    Expected sizes of constructs:

    • pSB1C3_ß-globin_YFP_hGH_rITR: ~4000 bp
    • pSB1C3_leftITR_pTERT: 2896 bp, ~640 bp

    The corresponding bands were cut out and Gel-Extraction was performed according to protocol.

    concentrations measured via NanoDrop:

    • pSB1C3_ß-globin_YFP_hGH_rITR: 34.73 ng/µl
    • pSB1C3_leftITR_pTERT: 9.48 ng/µl


    Ligation:

    T4 ligation was performed according to protocol.

    Volumes used:

    • vector: 2,9 µl
    • insert: 5,1 µl


    Transformation:

    bacterial strain used: XL1bB

    antibiotic used for plate: chlorampenicol

    Transformation was performed according to protocol.
    Plate was prepared and put in 37°C room.

    Continuation: Digestion of pSB1C3_NLS (P324)

    Investigator: Patrick

    Freigem10 Pat 3008.JPG

    The gelextraction was performed with QIAEX II Gel Extraction Kit which should also allow to extract fragments smaller than 70 bp (at least 40 bp). According to the QIAGEN Gel Extraction Kit the lenght of the DNA fragment should be about 70 bp. Unfortunately the DNA fragment was blurred so a quite large piece had to be cut out.

    The extraction yielded 4,26 and 3,77 ng/µl (two measurements) but this sample also quite polluted. ... put into freezer

    CD biobrick production

    Investigator: Kira
    NA samples were diluted 1:100


    Ingredients CD sample
    5X Phusion HF buffer 10 µl
    10 mM dNTP mix1µl
    forward primer: O158 2,5µl
    reverse primer: O159 2,5 µl
    DNA Template0,5 µl
    DMSO 0 µl
    Phusion Polymerase0,5 µl
    H2O33µl
    Total volume50 µl


    PCR program:

    CyclesTemperatureTime
    98°C1
    8x98°C15"
    60°C25"
    72°C60"
    17x98°C15"
    65°C25"
    72°C60"
    1x72°C5'
    Hold 4°C


    Digestion of plasmid backbone:

    c (pSB1C3) = 151, 1 ng/ µl

    Components vector Volume/µL
    DNA 1 µg 6,0 µl
    BSA (100x) 0,2 µl
    Buffer no. 4 (10x) 2,0 µl
    Enzyme 1 XbaI 0,5 µl
    Enzyme 2 AgeI HF 0,5 µl
    H2O 10,8 µl
    Total volume 20


    incubation @ 37 C for approx. 2 h


    Digestion of PCR product:

    Components PCR product Volume/µL
    DNA 30,0 µl
    BSA (100x) 0,4 µl
    Buffer no. 4 (10x) 4,0 µl
    Enzyme 1 XbaI 1,5 µl
    Enzyme 2 AgeI HF 1,0 µl
    H2O 3,1 µl
    Total volume 40


    incubation @ 37 C for approx. 2 h

    1% agarose gel

    Ligation
    T4 ligase was used
    1 ul T4 Buffer
    1 ul T4 Ligase
    9 ul (6,9 ul vector+ 1,1 ul insert) DNA-mix

    incubation over-night @ 18C

    Repetition of test digestion of pSB1C3_Rep40_RC_Insert and pSB1C3_Rep68_RC_Insert.

    Investigator: Chris L.

    • P316 = pSB1C3_Rep40_ins clone 1
      c=302,22 ng/µl
    • P317 = pSB1C3_Rep40_ins clone 2
      c=274,37 ng/µl
    • P318 = pSB1C3_Rep68_ins clone 1
      c=491,69 ng/µl
    • P319 = pSB1C3_Rep68_ins clone 2
      c=498,71 ng/µl


    Test digestion:

    components volume of P316/µl volume of P317/µl volume of P318/µl volume of P319/µl
    DNA 1 1 1 1
    BSA (10x) 1 1 1 1
    Buffer 4 (10x) 1 1 1 1
    Enzyme XcmI 0,5 0,5 0,5 0,5
    H2O 6,5 6,5 6,5 6,5
    Total volume /µl10 10 10 10


    Incubation time: 1 h, Incubation temperature: 37° >br /> Preparation of gel:
    0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 120 Volt, running time: 45 minutes
    Freiburg10 pSB1C3 Rep40 ins and pSB1C3 Rep68 ins.png

    Comment: Test digestion looks good. Clones 2 of each plasmid (P317 and P319) were sent in for sequencing with VR2 Primer.

    Midi-Prep of pAAV_RC_insertparts clone1

    Investigators: Chris W.

    Comment: Midi-Preps of B208

    The Midi-Preps were performed according to the standard protocol yielding the following concentrations:

    plasmid-no. P325p326
    concentration (ng/µl)2481,04 2050,66


    FACS analysis, Seeding HT1080 and AAV293

    Investigators: Kerstin, Adrian

    • FACS analysis of Transduction from 28.08.2010
    • Seeding HT1080 cells for Transduction (31.08.2010): 5x 6-well plates, 270.000 cells per well
    • Seeding AAV293 cells for Transfection (01.09.2010): 10 plates, 300.000 cells per well

    Mini-Preps and test digestion of Loop insertion BioBricks

    Investigator: Anna

    Comment: Two clones of each ViralBrick were prepared and test digested.

    Mini-Preps:

    p331 p335p339p342p345
    ConstructpSB1C3_587_KO_BAP_clone3.1 pSB1C3_587_RGD_clone5.1 pSB1C3_453_His_clone7.1 pSB1C3_587_His_clone8.3 pSB1C3_587_KO_empty_clone10.1
    DNA-Concentration / µl65,08 94,29 73,4969,05 66,73

    Test digestion:

    Freiburg10 LoopInsertions Sal Bam.jpg

    1% Agarose gel: 1 g Agarose, 100 ml TAE, 6 µl Gelred
    Marker: Gene ruler ladder DNA mix, 5 µl

    Used enzymes: SalI, BamHI (in general, SDS has to be added because of the strong binding of enzymes to DNA!)

    Comment: Test digestion had no significant results. In addition, one sample was forgotten, it was not clear which one (description makes no sense). Test digestion has to be repeated.



    Repetition of subcloning Cap-insert into pAAV_RC

    Investigator: Stefan

    Comments: There are still problems with BspMI. It was assumed that longer digestion could improve the results, therefore digestion with BspMI will be performed overnight.
    P.S: Welcome back Hanna!



    Digestion of vector and insert:

    • Insert: pMA_RepCap Vector_SDM_InsPvuII clone 1 (P211)
    • Vector: pAAV_RC_ins-rep clone1 (P250)



    A two-step digestion was performed:

    1st step

    P211 / µl P250 / µl
    DNA8,5 2
    buffer 34,6 5
    Enzyme BsiWI1 1
    H2O32,4 38
    total volume46 46


    2nd step

    P211 / µl P250 / µl
    Mix obtained from step 1 46 46
    Enzyme BspMI4 4
    total volume50 50



    Digestion will be performed overnight and continued tomorrow.

    106. labday 31.08.2010

    Cloning ZEGFR:1907 and 6xHis_middlelinker into pCerulean

    Investigator: Hanna

    Comment: In order to fuse the His-Tag to VP2, the His-Tag-Middlelinker construct will be cloned into the pCerulean plasmid (contains CMV promoter and SV40 terminator) today. In the next step, VP2 will be cloned behind the His-Tag-Middlelinker.
    In addition to that the Affibody (ZEGFR:1907) will be also cloned into pCerulean (without linker). Also here, the next step will be fusing VP2 behind the this targeting molecule.


    Practical Cloning:

    • plasmid:
      • Vector: name: pCerulean; number: P273
      • Insert: name: pSB1C3_6xHis_middlelinker (P310) + pSB1C3_Zegfr:1907 (because P267 was empty, P285 was used! See lab day 23.08.!)
    • new vector name: pCerulean_6xHis_middlelinker + pCerulean_Zegfr:1907
    • buffer used: 4; Restriction-enzymes used: XbaI and PstI


    Comments: Because the P267 tube was empty (!), P285 was used which was prepared by Jessy - I don't know whether this is a trafo of the glycerol stock? Sequenced?

    Digestion

    components volume of pCerulean/µl volume of pSB1C3_6xHis_middlelinker/µl volume of pSB1C3_ZEGFR:1907
    DNA 3.6 16.2 11.5
    BSA (10x) 3 2.5 2
    Buffer 4 (10x) 3 2.5 2
    Enzyme 1 XbaI 1.5 1 1
    Enzyme 2 PstI 1.5 1 1
    H2O 17.4 1.8 2.5
    Total volume 30 25 20
    • Incubation: 1.5 h



    Agarose-Gel:


    0.8 g Agarose, 55 ml TAE (1.45 %), 3 µL GELRED, at 115 Volt, running time: 35 minutes

    Sample Sample/µl] Loading dye (6x)/µl Expected size 1 (Geneious)
    P285 20 µl 4 µl 210 bp
    P273 30 µl 6 µl ~ 3900 bp
    P310 25 µl 5 µl 86 bp


    • Marker: GeneRuler ladder mix
    Marker /µL Sample P285 /µl Sample P273 /µl Sample P310 /µl
    Lane 5.5 24 25 25


    Freiburg10 Digestion 6xHis middlelinker and Zegfr.JPG.gif

    Gel extraction


    Gel measurement:

    Sample Weight Volume Concentration
    P285 240 mg 20 µL 5.4 ng/µL
    P273 130 mg 20 µL 14.5 ng/µL
    P310 220 mg 20 µL 2.4 ng/µL


    Comment: The DNA concentrations were very bad, which could be due to the fact, that the 6xHis_middlelinker construct's size is just 86 bp and the affibody's size is just 210 bp...


    Ligation


    1. pCerulean_6xHis_middlelinker

    6xHis_middlelinker pCerulean
    Volume/µl 2.57 6.43


    2. pCerulean_Zegfr:1907

    Zegfr:1907 pCerulean
    Volume/µl 2.72 6.28


    Trafo


    Trafo was performed following the standard protocol. Cells: XL1b (60 µL), DNA amount per sample: 3 µL; cells were plated onto Kanamycin-plates.

    PCR of SV 40 terminator

    Investigator: Bea

    Comment: In order to obtain the SV40 poly adenylation site and clone it into the iGEM standard plasmid pSB1C3 a PCR needs to be performed with the pEGFP-C1 which contains the SV40 terminator.


    • Plasmid used for PCR: pEGFP-C1 (P230)
    • 1:1000 dilution of P230 has been prepared
    • Plasmid used as vector: pSB1C3_CFP1 (P51.2)


    PCR (was performed following the standard protocol)

    Ingredients Volume / µl
    5X Phusion HF buffer 10
    10 mM dNTP mix1
    forward primer: O160 2,5
    reverse primer: O1612,5
    DNA Template3
    Phusion Polymerase0,5
    H2O30,5
    Total volume50


    PCR program:

    PCR Programtemperature/ °CTime
    198 1min
    298 15s
    8x59 25s
    3 727s
    4 98 15s
    17x6725s
    5727s
    6x725min
    Hold4

    After the PCR was performed, the sample was loaded on a 1.5% agarose gel and run for 30 minutes. As it can be seen in the picture below the expected size of the PCR product (260bp) can be confirmed. The lower band corresponds to the added primers and does not seem to be an additional band.

    Freiburg10 PCR product SV40.jpg


    For cloning the obtained fragment into the pSB1C3 standard vector, the construct need to be digested with the same restriction enzymes as the PCR product will be cut with. In this case the primers were designed with XbaI overhang for the prefix, and with SpeI-NotI-PstI overhang for the suffix. Therefore the vector will be cut with XbaI and PstI. The sample were incubated at 37 C for approx. 1.5 h

    • Plasmid used: pSB1C3_CFP (P51.2) c=150ng/µL
    • Add BSA
    • XbaI and PstI were used


    Digestion of vector:

    Components vpSB1C3_CFP/µL
    DNA 8
    BSA (100x) 2
    Buffer no. 4 (10x) 2
    Enzyme 1 XbaI 1
    Enzyme 2 AgeI HF 1
    H2O 6
    Total volume 20


    • Incubation of the sample at 37°C
    • Incubation for 120 minutes


    After the PCR has been cut out of the gel and purified with the Qiagen Gel Extraction Kit obtaining a concentration of 45 ng/µl measured with the Nanodrop, the complete PCR product volume of 29 µL was digested with XbaI and PstI for 2 hours.

    Digestion of the PCR product SV40

    Components vPCR product/µL
    DNA 29,0 µl
    BSA (10x) 4,0 µl
    Buffer no. 4 (10x) 4,0 µl
    Enzyme 1 XbaI 1,0 µl
    Enzyme 2 AgeI HF 1,0 µl
    H2O 1,0 µl
    Total volume 40
    • Incubation of the sample at 37°C
    • Incubation for 120 minutes


    The digested PCR product SV40 was purifed with the PC Purification Kit and concentration was measured.

    • cSV40= 31,5 ng/µl
    • cpSB1C3= 7,74 ng/µL

    For T4 ligation of the two fragments, the needed volumes of insert (SV40) and vector (pSB1C3) were calculated with the labtools program. The total volume DNA (vector/insert) mix will be 8 µL.

    • vSV40= 0,67 µL
    • vpSB1C3= 7,33 µL
    • vT4 buffer= 1 µL
    • vt4 ligase= 1 µL

    The ligation reaction was carried out for 40 minutes at room temperature. The ligated plasmid was transformed into XL1-Blue cells and plated on agar plates containing the appropriate amount of chloramphenicol.

    Cloning of NLS into pCerulean_VP1up

    Investigator: Anissa

    Comment: NLS was already 2 times cut of the pSB1C3_NLS and purified by Patrick (NLS 1: of the 29.08.10, NLS 2: of the 30.08.10) . Now, pCerulean_VP1up will be digested and NLS will be ligated into it.


    • Digestion of pCerulean_VP1up (p303)

      components Vector
      DNA 2,6
      BSA (10x) 1,5
      Buffer 4 (10x)1,5
      Enzyme PstI 1
      Enzyme AgeI 1
      H2O7,4
      Total volume 15


    • Gel:
      A 1% gel was made and the sample was running 30 minutes.
      Anissa310810.png
      Afterwards a gelextraction according the qiagen-protocol was performed.

    • Ligation:
      components Volume /µl concentration /ng/µl
      NLS 2 insert of 30.08.10 1,51 3
      NLS 1 insert of 29.08.101,513
      pCerulean_VP1up6,49 29


    • Transfomation: was performed into XL1 blue on a kanamycin plate according the standard protocol.

    Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273)

    Investigator: Patrick
    P276 digestion (207 ng/µl): 14 µl DNA sample, 2 µl Buffer 4, 2 µl BSA, 1 µl XbaI, 1 µl PstI-HF.
    P273 digestion (419 ng/µl): 4 µl DNA sample, 1 µl Buffer 4, 1 µl BSA, 1 µl XbaI, 1 µl PstI-HF.
    Digestion time: 1 h 45 min.

    Expected size of the fragments:
    P276: 786 & 2058 bp
    P273: 756 & 3938 bp

    My dear friend, where is your initial comment? Nice gel :) Yours sincerely, Hanna :)

    see: http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/August_2010#Strategy_for_VP2_N-terminal_fusion_and_VP1_insertion_of_targeting_moelcules
    Haha, good idea! :)
    Freiburg10 3108pat2.JPG


    Gelextraction: Obviously there was something wrong with the gel or the sample. The marked bands were cut out and the gelextraction was performed acording to he standard protocol, yielding the following concentrations:
    P276 up: 12,6 ng/µl
    P276 down: 9,9 ng/µl
    P273: 37,7 ng/µl

    All of them were polluted.

    There were two bands at a level where no bands should be. P 276 looks also strange because accidentally 6,5 µl 1kb GeneRuler were pipetted into the sample. The expected bands are all blurred. Even the expected 756 bp fragment of P273 is not at the level it should be. To ensure i dont throw away the insert i cut out both bands from P276. Now tomorrow there will be a ligation and a transformation with two samples instead of one



    Sequencing results of pSB1C3_6xHis_middlelinker and pSB1C3_6xHis_mVenus

    Investigator: Hanna

    1st Comment: Sequencing of pSB1C3_6xHis_middlelinker looked good:


    Freiburg10 6xHis middlelinker.JPG

    2nd Comment: Sequencing of mVenus looked good. The His-Tag delivered 2 additional bp and the suffix 1 transition (see picture). This could be due to bad sequencing qualities at this region... but needs to be verified!

    Freiburg10 6xHis mVenus.JPG


    Contiuation: Repetition of subcloning Cap-insert into pAAV_RC

    Investigator: Stefan

    Comments: Overnight digestion does not look good, therefore a new approach will be performed today using different concentrations of spermidine (0,5mM, 2,5mM and 10mM) because in Oller et al (Biochemistry, 1991) it was suggested that this improves digestion with BspMI.

    A two-step digestion was performed:

    1st step

    P211 / µl P250 / µl
    DNA8,5 2
    buffer 34,6 5
    Enzyme BsiWI1 1
    H2O32,4 38
    total volume46 46


    • Incubation: 70 minutes; 55°C

    2nd step

    P211 / µl P250 / µl
    Mix obtained from step 1 46 46
    Enzyme BspMI4 4
    total volume50 50


    • Incubation: 2,5h ; 37°C

    Agarosegel
    0.4 g Agarose, 50 ml TAE (0,8 %), 3 µL GELRED, at 115 Volt



    Freiburg10 P250 without Spermidine.png
    Freiburg10 P250 with spermidine.png
































    Gel extraction
    Gel-Ex was performed according to protocol.
    NanoDrop:

    • pMA_RepCap-insert: c= 7,42 ng/µl
    • pAAV_RC_ins-rep: c=12,9 ng/µl


    T4 Ligation
    Volume insert: 3,43 µl
    Volume vector: 4,57 µl
    Ligation incubated for 50 minutes at room temperature.

    Transformation Trafo was preformed according to standard protocol using XL1blue cells on plates containing Ampicilin.

    2nd Repetition of test digestion of Loop insertion BioBricks

    Investigator: Achim, Anna

    Comment: First test digestion didn't work (see 31.08.10), this time a new approach was done with NotI.

    Test digestion:

    components Volume for each sample /µl
    DNA 10
    BSA (10x) 1,5
    Buffer 4 (10x) 1,5
    Enzyme NotI 0,5
    H2O 1,5
    Total volume /µl15


    Sample-No. 1+2 3+4 5+6 7+8 9+10 11+12 13+14 15+16 17+18 19+20 21-23 24+25 26+27 28+29 30 31
    Components 453 BAP 587 BAP 587 KO BAP 453 RGD 587 RGD 587 KO RGD 453 HIS 587 HIS 587 KO HIS 587 KO EMPTY 453 Z34C 587 Z34C 587 KO Z34C 587 KO Z34C SPACER pSb1C3_Bla (cut version) pSb1C3_Bla (uncut)
    Oligo 1O124: 10 O126: 10 O128: 10 O130: 10 O132: 10 O134: 10 O135: 10 O137: 10 O139: 10 O141: 10 O143: 10 O145: 10 O147: 10 O149: 10 --- ---
    Oligo 2 O125: 10 O127: 10 O129: 10 O131: 10 O133: 10 O151: 10 O136: 10 O138: 10 O140: 10 O142: 10 O144: 10 O146: 10 O148: 10 O150: 10 --- ---


    Incubation time: 1 h, Incubation temperature: 37°

    Preparation of gel:
    1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 45 minutes

    Freiburg10 LoopInsertions.jpg


    Expected fragment sizes /bp:
    pSB1C3: 2051 bp Bla: 905
    BAP: ~150
    RGD: ~ 130
    Z34C: ~ 200
    His: ~ 125

    The following clones were sent for sequencing: 2,3,5,7,9,11,13,16,17,19 and 25.

    Comment: Clones (see above) were sent for sequencing with VR2 Primer. In addition, 8 mL DYT medium was inoculated with each of the clones for preparation of glycerol stocks. For sequencing results go to labday 01.09.10.

    Midi-Prep of pAAV_RC & pHelper

    Investigators: Chris W.

    Comment: Midi-Preps of pHelper=P356 and pAAV_RC=P357

    The Midi-Preps were performed according to the standard protocol yielding the following concentrations:

    plasmid-no. P356P357
    concentration (ng/µl)1068,0 1274.80


    Transformation of Biobrick CD

    Investigator: Kira
    Transformation was performed according to the standard protocol.

    • Retrieved from "http://2010.igem.org/Team:Freiburg_Bioware/testpage"