Team:Freiburg Bioware/NoteBook/Labjournal/October2

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[https://2010.igem.org/Team:Freiburg_Bioware/NoteBook => Back to Notebook overview]<br><br>
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<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September">September part 1 (labday 107 - 123)</a></li>
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September">September part 1 (labday 107 - 123)</a></li>
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2">September part 2 (labday 124 - 135)</a></li>
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2">September part 2 (labday 124 - 135)</a></li>
-
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October part 1 (labday 136 - 145 )</a></li>
+
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October part 1 (labday 136 - 149 )</a></li>
-
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2">October part 2 (labday 146 - 155 )</a></li>
+
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2">October part 2 (labday 150 - 166 )</a></li>
-
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October3">October part 3 (labday 156 - 166 )</a></li>
+
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November  (labday 167 - 170 )</a></li>
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November  (labday 167 - 170 )</a></li>
-
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture </a></li>
+
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture</a></li>
</ul>
</ul>
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-
===<p style="font-size:17px; background-color:#00dd77;">146. labday 11.10.2010</p>===
+
===<p style="font-size:17px; background-color:#00dd77;">150. labday 15.10.2010</p>===
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Colony PCR of Cloning VP2 Fusion and Super constructs into pSB1C3</b></p>====
+
====<p style="font-size:15px; background-color:#66bbff;">Cloning CFP (from P666: PSB1C3_CFP) into pSB1C3_leftITR_CMV_beta-globin (P729)</p>====
-
<b>Investigator: Achim, Hanna</b><br>
+
'''Investigator Patrick <br>
-
<br/>
+
Digestions, 2 h 10 minutes, 37 °C:
-
<b>Comment:</b> Because yesterday's cloning didn't deliver a good separartion of the expected gel bands. Nevertheless ligation and trafo was performed. In order to immediately find out, whether we received successful results a colony PCR will be performed. <br/>
+
*P666: 5 µl DNA, 2 µl BSA, 2 µl Buffer 4 (10x), 1 µl Xba, 1 µl PstI, 9 µl H2O
-
Two clones were picked from each plat. In addition to that a positive (pAAV_RC) and a negative control (pSB1C3_lITR) was prepared. <br/>
+
*P729: 4 µl DNA, 2 µl BSA, 2 µl Buffer 4 (10x), 1 µl SpeI, 1 µl PstI, 10 µl H2O
-
Used primer: 4200 rev and Cap3500 for. Expected fragment size: 885 bp. <br/>
+
-
PCR was performed following the standard protocol. <br/>
+
-
[[Image:Freiburg10 1110 testdigestionhanna1label.png|thumb|none|800px]]
+
Expected results for the 1% agarose gel:
 +
*P666: about 2100 and 750 bp
 +
*P729: about 3300 and 20 bp
-
[[Image:Freiburg10 1110 testdigestionhanna2label.png|thumb|none|800px]]
+
<br>
 +
[[Image:Freiburg10_1015pat_fertig.jpg|thumb|center|600px]]
 +
<br>
 +
<br>
 +
The gelextraction ...
 +
 
 +
P728: 11,8 ng/µl
 +
<br>
 +
P822: 34,6 ng/µl
 +
 
 +
... and following ligation (2,5 µl Insert, 5,5 µl vector, 1 µl T4 DNA Ligase, 1 µl T4 DNA Ligase Buffer (10x), 40 minutes, RT) were performed according to the standard protocol. After the transformation (with XL1B) the cells were plated and put into the 37°C room.
 +
Two additional transformations were performed with ligations from Volker labeled: "ligation viral brick 453 empty" & "viral brick 587 empty". <br>
 +
 
 +
The following day the plates were checked for clones. Unfortunately there grew no clones on these two plates contrary to my plate with a a lot of clones.
 +
 
 +
====<p style="font-size:15px; background-color:#66bbff;">Midi-Prep</p>====
 +
 
 +
'''Investigator: Chris W. <br>'''
 +
<p style="font-size:13px; color:#003399;"> Midi-Prep of:</p><br/>
 +
pSB1C3_001_RC_IRCK_P5tataless clone 1 =P866 =B516<br/>
 +
pSB1C3_001_CMV_VP123_587-KO_Z34C_spacer clone2 =P867 =B526<br/>
 +
pSB1C3_001_CMV_VP123_587-KO_Z34C clone2 =P868 =B529<br/>
 +
pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_BAP clone 1 =P869 =B680<br/>
 +
pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_6xHis clone 1 =P870 =B200<br/>
-
All samples match the positive control!
 
-
<br/>
 
-
<b>To do:</b> Mini-Prep and sequencing.
 
-
<br/>
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Preparation of SDS-PAGE gel (10%)</b></p>====
 
-
<b>Investigator: Hanna</b><br>
 
-
<br/>
 
-
<b>Comment:</b> In order to perform a Western Blot of different virus capsids (with and without capsid-motifs), 2 10% SDS polyacrylamid gels were prepared. <br/>
 
-
<br/>
 
-
<b>Resolving gel: 15 mL</b>
 
-
<br/>
 
-
* H<sub>2</sub>O: 5.9 mL
 
-
* Acryl-bisacrylamide mix (30%): 5 mL
 
-
* Tris (1.5 M, pH 8.8): 3.8 mL
 
-
* SDS (10%): 0.15 mL
 
-
* Ammonium persulfate (10%): 0.15 mL
 
-
* TEMED: 0.006 mL
 
<br/>
<br/>
 +
The Midi-Preps were performed according to the standard protocol yielding the following concentrations:
-
<b>Stacking gel (5%): 5 mL </b>
+
{| border="1"
 +
| plasmid-no. || align="right" |P866|| align="right" |P867|| align="right" |P868|| align="right" |P869|| align="right" |P870
 +
|-
 +
| concentration (ng/µl)|| align="right" |899,80 || align="right" |954,46 || align="right" |406,97|| align="right" |1642,76|| align="right" |1585,12
 +
|}
 +
<br>
<br/>
<br/>
-
* H<sub>2</sub>O: 3.4 mL
+
====<p style="font-size:15px; background-color:#66bbff;">mini prep of several constructs</p>====
-
* Acryl-bisacrylamide mix (30%): 0.83 mL
+
'''Investigator: Kira <br/>
-
* Tris (1.5 M, pH 6.8): 0.63 mL
+
 
-
* SDS (10%): 0.05 mL
+
c(rep52_1)=299,04 ng/ul<br/>
-
* Ammonium persulfate (10%): 0.05 mL
+
c(rep52_2)=290,07 ng/ul<br/>
-
* TEMED: 0.005 mL
+
c(rep78_1)=142,32 ng/ul<br/>
 +
c(rep78_2)=175,36 ng/ul<br/>
 +
 
 +
====<p style="font-size:15px; background-color:#66bbff;">Cell culture</p>====
 +
'''Investigator: Kira <br/>
 +
 
 +
The cells are still alive. Medium was exchanged.--> RNA will be harvested tomorrow
 +
 
 +
====<p style="font-size:15px; background-color:#66bbFF;"><b>Mini-Prep and test digestion of pSB1C3_CD_SDM-PstI_hGH_rITR</b></p>====
 +
<b>Investigator: Stefan</b><br />
 +
 
 +
Glycerol stocks were prepared:<br>
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Colony PCR of p40_VP123_capins</b></p>====
 
-
<b>Investigator: Bea</b><br>
 
-
<p style="color:#66bbff;"><i>Comment</i>: Since the first attempt did not work,and no cells grew on the plate and the same ligation was transformed again into BL-21 and a lot of clones grew on the plate, I decided to perform a colony PCR in order to check several colonies and to inoculate at the same day for a Midi-Prep. </p>
 
-
<b>Protocol:</b><br />
 
<ul>
<ul>
-
<li>Primer used: O162</li>
+
<li>B694 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 1</li>
-
<li>Primer used: O38</li>
+
<li>B695 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 2</li>
 +
 
 +
</ul><br>
 +
 
 +
Mini-Prep was performed according to standard protocol:<br>
 +
 
 +
<ul>
 +
<li>P875 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 1 c = 73,1 ng/µl</li>
 +
<li>P876 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 2 c = 78,3 ng/µl</li>
</ul>
</ul>
-
[[Image: Freiburg10_ColonyPCR_p40vp123_1110.PNG|thumb|center|600px]]
+
 
 +
Test digestion:<br>
 +
{| border="1"
 +
| align="left" | '''Components''' ||align="left"| <b>P875 + P876 / µl</b>
 +
|-
 +
| align="left" | DNA  ||align="left"| 4
 +
|-
 +
| align="left" | Buffer 4 ||align="left"| 1
 +
|-
 +
| align="left" | BSA (10x)  ||align="left"| 1
 +
|-
 +
| align="left" | XbaI ||align="left"| 0,3
 +
|-
 +
| align="left" | AgeI ||align="left"| 0,3
 +
|-
 +
| align="left" | H<sub>2</sub>O ||align="left"| 3,4
 +
|-
 +
| align="left" | '''Total volume''' ||align="left"| <b>10</b> 
 +
|}
 +
 
 +
Gel:<br>
 +
0,5g agarose, 50 ml TAE (1%), 3 µl GELRED, 115 Volt, running time ~50 minutes<br>
 +
 
 +
[[Image:Freiburg10 td151010.jpg|550px|thumb|center]]
 +
<p style="color:#66bbff;"><i>Comment</i>: Test digestion looks allright, cloning will be continued using P876.</p>
 +
 
 +
===<p style="font-size:17px; background-color:#00dd77;">151. labday 16.10.2010</p>===
 +
====<p style="font-size:15px; background-color:#66bbff;">Biobrick assembly: pSB1C3_lITR_CMV_ß-globin_CD_hGH_rITR and pSB1C3_lITR_phTERT_ß-globin_CD_hGH_rITR </p>====
 +
 
 +
'''Investigator: Achim'''
 +
 
 +
'''Plasmids:'''
 +
*P729: pSB1C3_lITR_CMV_ß-Globin
 +
**c= 243.4 ng/µl
 +
*P730: pSB1C3_lITR_pHTERT_ß-Globin
 +
**c= 81.1 ng/µl
 +
*P876: pSB1C3_CD_SDM-PstI_hGH_rITR
 +
**c= 78.3 ng/µl
 +
'''Digestion:'''
 +
 
 +
{| border="1"
 +
| <b>components</b>  || align="right" |<b>I1 (P729)</b> || align="right" |<b>I2 (P730)</b> || align="right" |<b>V (P876)
 +
|-
 +
| DNA  ||  align="right" |6 ||  align="right" |14||  align="right" |14
 +
|-
 +
| BSA (10x) ||  align="right" |2 ||  align="right" |2||  align="right" |2
 +
|-
 +
| Buffer 4 (10x)||  align="right" |2 ||  align="right" |2 ||  align="right" |2
 +
|-
 +
|Enzyme EcoI||  align="right" |1||  align="right" |1 ||  align="right" |1
 +
|-
 +
|Enzyme XbaI||  align="right" |-||  align="right" |- ||  align="right" |1
 +
|-
 +
|Enzyme SpeI||  align="right" |1||  align="right" |1 ||  align="right" |-
 +
|-
 +
|H2O||  align="right" |8||  align="right" |- ||  align="right" |-
 +
|-
 +
|'''Total '''||  align="right" | 20||  align="right" | 20||  align="right" | 20
 +
|}
 +
 
 +
Digestion: 2h, 37°C
 +
 
 +
'''Prep. gel:'''
 +
*0,8%, run for 45 min
 +
 
 +
[[Image:Freiburg10 1610 bba.png|thumb|none|400px|Expected Bands: I1: 1335, I2: 1138, V: 4000]]
 +
 
 +
*Corresponding bands were cut out
 +
 
 +
'''Gel ex.'''
 +
 
 +
*Nanodrop concentrations:
 +
**I1: 37.54 ng/µl
 +
**I2: 25.38 ng/µl
 +
**V: 26.47 ng/µl
 +
 
 +
'''Ligation:'''
 +
{| border="1"
 +
|ligation name || align="right" |I1 + V|| align="right" |I2 + V
 +
|-
 +
|volume of vector || align="right" |4.69|| align="right" |4.23
 +
|-
 +
|volume of insert|| align="right" |3.31|| align="right" |3.77
 +
|-
 +
|T4 ligase buffer (10x)|| align="right" |1|| align="right" |1
 +
|-
 +
|T4 ligase || align="right" |1|| align="right" |1
 +
|-
 +
|}
 +
 
 +
*Ligation @ RT for 40 min
 +
 
 +
'''Trafo:'''
 +
*Done by Kira
 +
 
 +
 
 +
<br>
 +
 
 +
====<p style="font-size:15px; background-color:#66bbff;">Mini-prep of mutual pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI</p>====
 +
 
 +
'''Investigator Patrick
 +
<br>
 +
 
 +
Yielded concentrations & given numbers:
 +
*pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 1:  208,4 ng/µl , P877 / B696
 +
*pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 2:  251,6 ng/µl , P878 / B696
 +
*pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 3:  212,6 ng/µl , P889 / B696
 +
 +
 
 +
 
 +
<br>
 +
Test-digestion: 0,5 µl SpeI, 0,5 µl PstI, 3 µl DNA, 1 µl Buffer 4, 1 µl BSA, 4 µl H2O, 40 minutes, 37°C
 +
<br>
 +
Expected results: fragments with about 650 and 4900 bp
 +
<br>
 +
[[Image:Freiburg10_1016pat_test.jpg‎|thumb|center|600px]]
 +
<br>
 +
Obviously, this test digestion has to be repeated.
 +
 
 +
Preparations for tomorrow:
 +
*Mini-prep of 3 mutual pSB1C3_leftITR_CMV_beta-globin_CFP clones (have to be picked from the plate)
 +
*Midi-prep of pHelper
 +
*Midi-prep of B689:pSB1C3_lITR_CMV_betaglobin_mGMK_TK30_SDM-PstI_hGH_rITR clone 2
 +
 
 +
====<p style="font-size:15px; background-color:#66bbff;">Biobrick assembly of Rep78 and Rep52</p>====
 +
 
 +
'''Investigator: Kira <br />
 +
'''Comment:''' After replacing the mutated Rep parts by the ordered Rep parts, PCR amplification has to be done in order to produce a biobrick.
 +
PCR program:
<br />
<br />
-
The PCR products were loaded on a 1% agarose gel. The results can be seen above in the gel picture: <br />
+
 
 +
c(Rep52)=299 ng/ul <br />
 +
c(Rep78)=175 ng/ul <br />
 +
 
 +
Rep52: praefix 094 & suffix 097 <br />
 +
Rep78: praefix 093 & suffix 097 <br />
 +
 
 +
 
 +
{| border="1"
 +
| components  || align="right" |volume in µl 
 +
|-
 +
| 5x Phusion HF buffer  ||  align="right" | 10
 +
|-
 +
| 10 mM dNTP mix ||  align="right" |1
 +
|-
 +
|primer_for (1:10 dilution)||  align="right" |  2,5
 +
|-
 +
|primer_rev (1:10 dilution)||  align="right" | 2,5 
 +
|-
 +
|DNA template (1:100)||  align="right" |  0,5
 +
|-
 +
|DMSO||  align="right" |  0,5
 +
|-
 +
|Phusion polymerase||  align="right" | 0,5
 +
|-
 +
|H<sub>2</sub>O||  align="right" | 32,5
 +
|-
 +
|'''Total volume (e.g. 50 µl)'''||  align="right" | 50
 +
|}
<br />
<br />
-
<b>Result: We can see two things: The cloning of p40 to VP123 worked quiet well AND the Robust PCR Kit which was used for the first time worked as well.</b>
+
 
 +
{| border="1"
 +
|Cycles||Temperature||Time
 +
|-
 +
|||98°C||30 sec
 +
|-
 +
|10x||98°C||15 sec
 +
|-
 +
|||63°C||25 sec
 +
|-
 +
|||72°C||32 sec
 +
|-
 +
|20x||98°C||15 sec
 +
|-
 +
|||66°C||25 sec
 +
|-
 +
|||72°C||32 sec
 +
|-
 +
|1x||72°C||5 min
 +
|-
 +
|Hold 4°C
 +
|}
 +
<br>
 +
1% agarose gel <br />
 +
[[Image:Freiburg10_Rep78&Rep52_PCR.jpg]]
 +
 
 +
Digestion of plasmid backbone:
 +
 
 +
pSB1C3_001 is used as backbone
 +
 
 +
{| border="1"
 +
| align="left" | '''Components''' ||align="left"| <b>vector</b> Volume/µL
 +
|-
 +
| align="left" | DNA  ||align="left"| 3,5 µl
 +
|-
 +
| align="left" | BSA (10x) ||align="left"| 2 µl
 +
|-
 +
| align="left" | Buffer no. 4 (10x) ||align="left"| 2,0 µl
 +
|-
 +
| align="left" | Enzyme 1 EcoRI-HF ||align="left"| 0,5 µl
 +
|-
 +
| align="left" | Enzyme 2 SpeI  ||align="left"| 1,0 µl
 +
|-
 +
| align="left" | H<sub>2</sub>O ||align="left"| 15 µl
 +
|-
 +
| align="left" | '''Total volume''' ||align="left"| <b>25</b>
 +
|}
<br />
<br />
-
[[Image: Freiburg10_ColonyPCR_p40VP123.png|thumb|center|400px]]
+
 
 +
incubation @ 37 C for approx. 2 h
 +
 
 +
1% agarose gel <br />
 +
[[Image:Freiburg10_digestion_pSB1C31_16.10.jpg|thumb|center|600px]]
 +
 
 +
Digestion of PCR product:
 +
{| border="1"
 +
| align="left" | '''Components''' ||align="left"| <b>PCR product</b> Volume/µL
 +
|-
 +
| align="left" | DNA  ||align="left"| 35,0 µl
 +
|-
 +
| align="left" | BSA (100x) ||align="left"| 0,45 µl
 +
|-
 +
| align="left" | Buffer no. 4  ||align="left"| 4,5 µl
 +
|-
 +
| align="left" | Enzyme 1 EcoRI-HF ||align="left"| 1,5 µl
 +
|-
 +
| align="left" | Enzyme 2 SpeI  ||align="left"|2,0 µl
 +
|-
 +
| align="left" | H<sub>2</sub>O ||align="left"| 1,5 µl
 +
|-
 +
| align="left" | '''Total volume''' ||align="left"| <b>45</b> 
 +
|}
<br />
<br />
 +
incubation @ 37 C for approx. 2 h <br />
 +
 +
T4 ligation for 40 min <br />
 +
Transformation according to the standard protocol <br />
 +
 +
====<p style="font-size:15px; background-color:#66bbff;">RNA harvesting</p>====
 +
 +
'''Investigator: Kira <br />
 +
After transfection, the cells were incubated for 48 hours. Today, the cells will be harvested and RNA extracted, in order to perform RT-PCR and an additional PCR for evaluation of promoter activity.<br />
 +
 +
The transfected cells were trypsinised and centrifuged for 2 min. The supernatant was discarded and pellet washed 2x with PBS. RNeasy Kit [Qiagen] was used for RNA extraction according to the manufacturer protocol. <br />
 +
 +
c(CMV)= 335,69 ng/ul<br />
 +
c(P40)= 857,92 ng/ul<br />
 +
c(AAV_RC)= 760,21 ng/ul<br />
 +
 +
===<p style="font-size:17px; background-color:#00dd77;">152. labday 17.10.2010</p>===
 +
====<p style="font-size:15px; background-color:#66bbff;"><b>Test digestion of pSB1C3_lITR_CMV_ß-globin_CFP</b></p>====
 +
<b>Investigator: Anna</b><br>
 +
 +
Vector name:<br />
 +
pSB1C3_lITR_CMV_betaglobin_CFP_cl1 (P880): c = 452,67 ng/µl<br />
 +
pSB1C3_lITR_CMV_betaglobin_CFP_cl2 (P881): c = 288,88 ng/µl<br />
 +
pSB1C3_lITR_CMV_betaglobin_CFP_cl3 (P882): c = 288,36 ng/µl<br />
 +
 +
<b>Test Digestion:</b>
 +
<br />
 +
{| border="1"
 +
| <b>components</b>  || align="right" |<b>volume P880 - P882  /µl</b> || align="right" |<b>volume P434  /µl </b>
 +
|-
 +
| DNA  ||  align="right" |2 ||  align="right" |2
 +
|-
 +
| BSA (10x) ||  align="right" |1 ||  align="right" |1
 +
|-
 +
| Buffer 4 (10x)||  align="right" |1 ||  align="right" |1
 +
|-
 +
|Enzyme NgoMIV||  align="right" |0,3||  align="right" |0,3
 +
|-
 +
|Enzyme AgeI||  align="right" |0,3||  align="right" |0,3
 +
|-
 +
|H2O||  align="right" |5,4||  align="right" |5,4
 +
|-
 +
|'''Total volume'''||  align="right" | 10||  align="right" | 10
 +
|}
 +
 +
<br />
 +
<b>Gel:</b><br />
 +
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt<br />
 +
 +
 +
<br/>
 +
[[Image:Freiburg10_Test digestion of pSB1C3_lITR_CMV_ß-globin_CFP.jpg|thumb|center|350px|]]<br/>
-
====<p style="font-size:15px; background-color:#66bbff;"><b>cloning of lITR_CMV_betaglobin and lITR_phTERT_betaglobin into pSB1C3_CD</b></p>====
+
====<p style="font-size:15px; background-color:#66bbff;"><b>Cloning of hGH_rITR into pSB1C3_lITR_CMV_betaglobin_CFP</b></p>====
<b>Investigator: Stefan</b><br>
<b>Investigator: Stefan</b><br>
-
<p style="font-size:13px; color:red;">Comment: To produce another GOI for testing in cell culture, the cytosine deaminase needs to be assembled with lITR_promotor_betaglobin. In the next step hgH_rITR needs to be added.</p><br />
+
<p style="font-size:13px; color:red;">Cloning of our last GOI!</p><br />
Vector name:<br />
Vector name:<br />
-
pSB1C3_CD clone 1 (P???)<br />
+
pSB1C3_lITR_CMV_betaglobin_CFP cl 1-3 (P880-P882)<br />
-
pSB1C3_CD clone 2 (P???)<br />
+
Insert name:<br />
Insert name:<br />
-
pSB1C3_lITR_CMV_beta-globin (P729)<br />
+
pSB1C3_hGH_rITR (P728)<br />
-
pSB1C3_lITR_phTERT_beta-globin (P730)<br />
+
<b>Digestion:</b><br /><br />
<b>Digestion:</b><br /><br />
Line 111: Line 415:
<br />
<br />
{| border="1"
{| border="1"
-
| <b>components</b>  || align="right" |<b>volume CD clone 1 + 2 /µl</b> || align="right" |<b>volume P729  /µl </b>||<b>volume P730 /µl</b>  
+
| <b>components</b>  || align="right" |<b>volume P880 - P882 /µl</b> || align="right" |<b>volume P728 /µl </b>
|-
|-
-
| DNA  ||  align="right" |6 ||  align="right" |14||  align="right" |6
+
| DNA  ||  align="right" |3 ||  align="right" |8
|-
|-
-
| BSA (10x) ||  align="right" |2 ||  align="right" |2||  align="right" |2
+
| BSA (10x) ||  align="right" |2 ||  align="right" |2
|-
|-
-
| Buffer 4 (10x)||  align="right" |2 ||  align="right" |2||  align="right" |2
+
| Buffer 4 (10x)||  align="right" |2 ||  align="right" |2
|-
|-
-
|Enzyme EcoI||  align="right" |1||  align="right" |1 ||  align="right" |1  
+
|Enzyme PstI||  align="right" |1||  align="right" |1  
|-
|-
-
|Enzyme XbaI||  align="right" |1||  align="right" |- ||  align="right" |-
+
|Enzyme XbaI||  align="right" |-||  align="right" |1
|-
|-
-
|Enzyme SpeI||  align="right" |-||  align="right" |1||  align="right" |1
+
|Enzyme SpeI||  align="right" |1||  align="right" |-
|-
|-
-
|H2O||  align="right" |8||  align="right" |- ||  align="right" |8
+
|H2O||  align="right" |11||  align="right" |4
|-
|-
-
|'''Total volume (e.g. 15,20,25,30 µl)'''||  align="right" | 20||  align="right" | 20||  align="right" | 20
+
|'''Total volume (e.g. 15,20,25,30 µl)'''||  align="right" | 20||  align="right" | 20
|}
|}
Line 136: Line 440:
<br/>
<br/>
-
[[Image:Freiburg10 cloning 111010.jpg|550px|]]<br/>
+
[[Image:Freiburg10 171010.jpg|550px|thumb|center]]<br/>
-
 
+
-
<br/>
+
-
 
+
-
<p style="font-size:13px; color:red;"> CD clone 1 yielded to bands around 2500 bp to 3000 bp. Since the vector was cut only using EcoRI and SpeI, it was expected to be linearized, not to be cut into two fragments this size. Therefore, this sample was discarded and cloning was continued using CD clone 2. </p><br />
+
 +
<p style="font-size:13px; color:red;">Test digestion of all constructs looked alright, therefore, cloning was continued using P881 only.</p><br />
 +
<br/>
<b>Gel extraction</b>: <br>
<b>Gel extraction</b>: <br>
Was performed according to protocol.
Was performed according to protocol.
Line 149: Line 451:
<b>T4 Ligation</b>: <br>
<b>T4 Ligation</b>: <br>
{| border="1"
{| border="1"
-
|ligation name || align="right" |729 + CD cl2|| align="right" |730 + CD cl2
+
|ligation name || align="right" |728 + 881
|-
|-
-
|volume of vector || align="right" |3,67|| align="right" |2,82
+
|volume of vector || align="right" |2,68
|-
|-
-
|volume of insert|| align="right" |4,33|| align="right" |5,18
+
|volume of insert|| align="right" |5,32
|-
|-
-
|T4 ligase buffer (10x)|| align="right" |1|| align="right" |1
+
|T4 ligase buffer (10x)|| align="right" |1
|-
|-
-
|T4 ligase || align="right" |1|| align="right" |1
+
|T4 ligase || align="right" |1
|-
|-
|}
|}
Line 163: Line 465:
<br>
<br>
<b>Transformation</b>: <br>
<b>Transformation</b>: <br>
-
Was performed according to standard protocol using BL21 cells.
+
Transformation was performed according to standard protocol using BL21 cells.
<br/>
<br/>
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Seeding HT1080 and A431 for testing different concentrations of ganciclovir by MTT-Assay</b></p>====
+
====<p style="font-size:15px; background-color:#66bbff;"><b>RT-PCR</b></p>====
-
<b>Investigator: Kerstin, Anissa</b><br>
+
<b>Investigator: Kira</b><br>
 +
For further experiments, RNA has to be translated into cDNA. The PCR was performed according to the manufacturer protocol.
-
*Seeding of 4x 96-well plates:
+
===<p style="font-size:17px; background-color:#00dd77;">153. labday 18.10.2010</p>===
 +
====<p style="font-size:15px; background-color:#66bbFF;"><b>quantitative real-time PCR for detection of virus titer</b></p>====
 +
'''Investigator: Achim <br>
-
*Transduction plan (12.10.2010):
+
*qPCR of harvested virus particles to determine the virus titers of our different constructs
-
[[Image:Freiburg10 Transductionplan1 11.10.2010.jpg|700px|]]<br/>
+
*Total number of samples: 58
-
[[Image:Freiburg10 Transductionplan2 11.10.2010.jpg|700px|]]<br/>
+
====<p style="font-size:15px; background-color:#66bbFF;"><b>Test-digestion of pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 1, 2 and 3 (P877, 878 and 879) </b></p>====
 +
'''Investigator Patrick <br>
-
[[Image:Freiburg10 Transductionplan3 11.10.2010.jpg|700px|]]<br/>
+
Check the plasmid for leftITR:
 +
0,5 µl EcoRI, 1 µl BstEII, 7 µl DNA, 2 µl Buffer 4, 2 µl BSA, 7,5 µl H2O, 45 minutes 37°C, 45 minutes 60°C
 +
<br>
 +
<br>
 +
Check the plasmid for hGH_rITR and ... :
 +
0,5 µl AgeI, 0,5 µl PstI, 3 µl DNA, 1 µl BSA, 1 µl Buffer 4, 4 µl H2O, 70 minutes 37°C
-
[[Image:Freiburg10 Transductionplan4 11.10.2010.jpg|700px|]]<br/>
+
Expected results:
 +
* leftITR: about 190 bp
 +
* hGH_rITR: about 670 bp
-
====<p style="font-size:15px; background-color:#66bbff;"><b>FACS-Analysis</b></p>====
+
<br>
-
<b>Investigator: Kerstin</b><br>
+
Unfortunately the digestions had to be reapeated because i didnt switch on the current so the samples and especially the 1kb GeneRuler marker diffused.
 +
<br>
 +
<br>
 +
The second run: see above <br>
 +
[[Image:Freiburg10_1019pat_test1bfertig.jpg]]
 +
<br>
 +
PstI or BstEII seems to work not properly
-
*Results Transduction
+
====<p style="font-size:15px; background-color:#66bbFF;"><b>MTT Assay </b></p>====
 +
'''Investigator Kerstin, Anissa <br>
 +
[[Image:Freiburg10 transductionplan 18.10..jpg]]
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Preparation of the ELISA</b></p>====
+
[[Image:Freiburg10 transductionplan1 18.10..jpg|thumb|center|920px]]
-
<b>Investigator: Volker</b><br>
+
 +
[[Image:Freiburg10 transductionplan2 18.10..jpg|thumb|center|920px]]
-
The AAV particle standard that contains 3.6x10^9 viral particles was dissolved in 500µl as described in the protocol of the Progen AAV Titration ELISA and a absorption spectrum was measured.
+
'''Results:'''
-
These purified and concentrated viral particles could be used for biophysical measurements, there for the possibility to detect the viral particles by absorption was interesting for us.
+
 
 +
[[Image:Freiburg10 Results of MTTAssay 18 10 10 2day 1pic.jpg|thumb|center|920px]]
 +
 
 +
[[Image:Freiburg10 Results of MTTAssay 18 10 10 2day 3pic.jpg|thumb|center|920px]]
 +
 
 +
[[Image:Freiburg10 Results of MTTAssay 18 10 10 2day 2pic.jpg|thumb|center|920px]]
 +
 
 +
====<p style="font-size:15px; background-color:#66bbFF;"><b>Mini-Prep and test digestion of several constructs</b></p>====
 +
<b>Investigator: Jessica</b><br />
 +
 
 +
Glycerol stocks were prepared:<br>
 +
 
 +
<ul>
 +
<li>B702 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 1</li>
 +
<li>B703 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 2</li>
 +
<li>B704 = pSB1C3_001_VP3 clone 1</li>
 +
<li>B705 = pSB1C3_001_VP3 clone 2</li>
 +
<li>B706 = pSB1C3_001_VP2 clone 1</li>
 +
<li>B707 = pSB1C3_001_VP2 clone 2</li>
 +
<li>B708 = pSB1C3_001_Rep78 clone 1</li>
 +
<li>B709 = pSB1C3_001_Rep78 clone 2</li>
 +
<li>B710 = pSB1C3_001_Rep52 clone 1</li>
 +
<li>B711 = pSB1C3_001_Rep52 clone 2</li>
 +
<li>B712 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 1</li>
 +
<li>B713 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 2</li>
 +
<li>B714 = pSB1C3_001_VP1 clone 1</li>
 +
<li>B715 = pSB1C3_001_VP1 clone 2</li>
 +
 
 +
</ul><br>
 +
 
 +
Mini-Prep was performed according to standard protocol:<br>
 +
 
 +
<ul>
 +
<li>P886 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 1 c= 232,2ng/µl</li>
 +
<li>P887 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 2 c= 186,2ng/µl</li>
 +
<li>P888 = pSB1C3_001_VP3 clone 1 c= 300,8ng/µl</li>
 +
<li>P889 = pSB1C3_001_VP3 clone 2 c= 284,4ng/µl</li>
 +
<li>P890 = pSB1C3_001_VP2 clone 1 c= 298,3ng/µl</li>
 +
<li>P891 = pSB1C3_001_VP2 clone 2 c= 299,9ng/µl</li>
 +
<li>P892 = pSB1C3_001_Rep78 clone 1 c= 143,9ng/µl</li>
 +
<li>P893 = pSB1C3_001_Rep78 clone 2 c= 163,4ng/µl</li>
 +
<li>P894 = pSB1C3_001_Rep52 clone 1 c= 166,6ng/µl</li>
 +
<li>P895 = pSB1C3_001_Rep52 clone 2 c= 181,6ng/µl</li>
 +
<li>P896 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 1 c= 250,5ng/µl</li>
 +
<li>P897 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 2 c= 173,4ng/µl</li>
 +
<li>P898 = pSB1C3_001_VP1 clone 1 c= 272,7ng/µl</li>
 +
<li>P899 = pSB1C3_001_VP1 clone 2 c= 294,8ng/µl</li>
 +
<li>P900 = pSB1C3_hGH_rITR (from B160) c= 136,7ng/µl</li>
 +
</ul>
 +
 +
Test digestion:<br>
 +
{| border="1"
 +
| align="left" | '''Components''' ||align="left"| <b>P886,887,892,893,894,895,896,897 / µl</b>||align="left"| <b>P888,889,890,891898,899 / µl</b>
 +
|-
 +
| align="left" | DNA  ||align="left"| 1,5||align="left"| 1,5
 +
|-
 +
| align="left" | Buffer  ||align="left"| (4) 1||align="left"| (2) 1
 +
|-
 +
| align="left" | BSA (10x)  ||align="left"| 1||align="left"| 1
 +
|-
 +
| align="left" | NgoMIV ||align="left"| 0,4 ||align="left"| -
 +
|-
 +
| align="left" | XbaI ||align="left"| 0,4 ||align="left"| -
 +
|-
 +
| align="left" | PstI ||align="left"| - ||align="left"| 0,6
 +
|-
 +
| align="left" | XcmI ||align="left"| - ||align="left"| 0,4
 +
|-
 +
| align="left" | H<sub>2</sub>O ||align="left"| 4,5||align="left"| 4,5
 +
|-
 +
| align="left" | '''Total volume''' ||align="left"| <b>10</b>  ||align="left"| <b>10</b>
 +
|}
 +
 
 +
Gel:<br>
 +
1,0g agarose, 100 ml TAE (1%), 6 µl GELRED,  Volt, running time  minutes<br>
 +
 
 +
[[Image:Freiburg10 test digestion 886-899.jpg|thumb|center|800px]]
 +
<p style="color:#66bbff;"><i>Comment</i>: Rep 52/78 will be checked,pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR and pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR will be sequenced</p>
 +
 
 +
====<p style="font-size:15px; background-color:#66bbff;"><b>PCR of mGMK and SR39</b></p>====
 +
<b>Investigator: Anna</b><br>
 +
 
 +
 
 +
Plasmids:<br />
 +
pSB1C3_mGMK_TK30_SDM-PstI clone 2(P804)<br />
 +
pSB1C3_mGMK_sr39 clone 1(P860)<br />
 +
 
 +
Oligos:<br />
 +
O193: pTK30_for<br />
 +
O81: pmgmk_tk30_suffix_RFC25_rev
 +
<br />
 +
 
 +
 
 +
<b>PCR Mix:</b>
 +
 
 +
{| border="1"
 +
| <b>Components</b>  || align="right" |<b>Volume  /µl</b>
 +
|-
 +
| Phusion Buffer  ||  align="right" |10
 +
|-
 +
| dNTP ||  align="right" |1
 +
|-
 +
|Primer_for||  align="right" |2,5
 +
|-
 +
|Primer_rev||  align="right" |2,5
 +
|-
 +
|DNA template||  align="right" |1
 +
|-
 +
|H2O||  align="right" |32,5
 +
|-
 +
|'''Total volume'''||  align="right" | 50
 +
|}
 +
<br />
 +
<b>PCR Program:</b>
 +
 
 +
{| border="1"
 +
|Cycles||Temperature||Time
 +
|-
 +
|||98°C||60 sec
 +
|-
 +
|||98°C||15 sec
 +
|-
 +
|8x||52°C||25 sec
 +
|-
 +
|||72°C||25 sec
 +
|-
 +
|||98°C||15 sec
 +
|-
 +
|17x||67°C||25 sec
 +
|-
 +
|||72°C||25 sec
 +
|-
 +
|1x||72°C||5 min
 +
|-
 +
|Hold 4°C
 +
|}
 +
 
 +
<br />
 +
<b>Gel:</b><br />
 +
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt<br />
-
The Spectrum measured in the NanoDrop is the following:
 
-
[[Image:Freiburg10_Absorption spectrum of the AAV particle standard.jpeg|thumb|center|800px]]
 
-
===<p style="font-size:17px; background-color:#00dd77;">147. labday 12.10.2010</p>===
 
-
====<p style="font-size:15px; background-color:#66bbff;"><b>Concentration of virus stock for Western Blot analysis</b></p>====
 
-
<b>Investigator: Hanna </b> <br/>
 
<br/>
<br/>
-
For the first Western Blot try, we investigate whether is is enough to concentrate the virus stock (from cell supernatant) and then to simply load it onto a SDS-PAGE gel and perfom immuno-staining. <br/>
+
[[Image:|550px|]]<br/>
-
For this purpose 8 mL virus stock were concentrated via amicon filtration. <br/>
+
 
-
* First, amicon filter was washed with 4 mL PBS.
+
===<p style="font-size:17px; background-color:#00dd77;">154. labday 19.10.2010</p>===
-
* Then 4 mL virus stock was loaded onto the filter and centrifuged at 3000 rpm for 15 minutes.
+
 
-
* Flow-through was discarded and additional virus stock was loaded, centrifuged at 3000 rpm for 25 minutes.
+
====<p style="font-size:15px; background-color:#66bbff;">Midi-Prep</p>====
-
* Flow-through was discarded, remaining solution was re-suspended and additional virus stock was added. Amicon filter was centrifuged at 3000 rpm for 50 minutes.
+
 
-
* Filter was washed with PBS two more times.
+
'''Investigator: Chris W. <br>'''
-
* 2 x 20 µL protein solution was taken and mixed with Laemmli buffer and stored over night @ 4°C.
+
<p style="font-size:13px; color:#003399;"> Midi-Prep of:</p><br/>
 +
pSB1C3_lITR_CMV_betaglobin_mVenus_hGH_rITR clone1 =P901 =B200<br/>
 +
pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 2 =P902 =B697<br/>
 +
 
 +
 
 +
 
 +
 
<br/>
<br/>
-
<b>To do:</b> SDS-PAGE and Western Blot tomorrow. <br/>
+
The Midi-Preps were performed according to the standard protocol yielding the following concentrations:
-
Perfomance of another try by harvesting viruses of the cell pellet with RIPA buffer & freeze/ thaw. <br/>
+
-
Western Blot of unmodified, and modified capsids (N-terminal fusion and VP1 insertion with CFP ref. mVenus) --> size shift should be detectable.<br/>
+
-
===<p style="font-size:17px; background-color:#00dd77;">148. labday 13.10.2010</p>===
+
{| border="1"
 +
| plasmid-no. || align="right" |P901|| align="right" |P902
 +
|-
 +
| concentration (ng/µl)|| align="right" |1563,63 || align="right" |1348,26
 +
|}
 +
<br>
 +
<br/>
 +
====<p style="font-size:15px; background-color:#66bbff;"><b>Continuation of PCR of mGMK and SR39</b></p>====
 +
<b>Investigator: Jessica</b><br>
-
===<p style="font-size:17px; background-color:#00dd77;">149. labday 14.10.2010</p>===
+
* vector (P320) and PCR product was digested<br>
-
===<p style="font-size:17px; background-color:#00dd77;">150. labday 15.10.2010</p>===
+
{| border="1"
 +
| align="left" | '''Components''' ||align="left"| <b>P320 / µl</b>||align="left"| <b>PCR product P804 and P860 / µl</b>
 +
|-
 +
| align="left" | DNA  ||align="left"| 1,5||align="left"| 20
 +
|-
 +
| align="left" | Buffer  ||align="left"| 2||align="left"| 3
 +
|-
 +
| align="left" | BSA (10x)  ||align="left"| 2||align="left"| 3
 +
|-
 +
| align="left" | AgeI ||align="left"| 1 ||align="left"| 1,5
 +
|-
 +
| align="left" | XbaI ||align="left"| 1 ||align="left"| 1,5
 +
|-
-
===<p style="font-size:17px; background-color:#00dd77;">151. labday 16.10.2010</p>===
+
| align="left" | H<sub>2</sub>O ||align="left"| 14,5||align="left"| 1
 +
|-
 +
| align="left" | '''Total volume''' ||align="left"| <b>20</b>  ||align="left"| <b>30</b>  
 +
|}
 +
[[Image:Freiburg10 pcr mGMK and SR39.jpg|thumb|center|800px]]
 +
<br>
 +
<br>
 +
'''Ligation'''
 +
* P320 c= 5,08 ng/µl
 +
* P804 c= 11,73 ng/µl
 +
* P860 c= 26,57 ng/µl
 +
<br>
 +
* P320 + P804: 4,93µl : 3,07µl
 +
* P320 + P860: 6,28µl : 1,72µl
 +
<br>
 +
'''Transformation with BL21 and Cm'''
-
===<p style="font-size:17px; background-color:#00dd77;">152. labday 17.10.2010</p>===
+
====<p style="font-size:15px; background-color:#66bbff;">Cloning of CMV into pSB1C3_001_VP1, pSB1C3_001_VP2 and pSB1C3_001_VP3</p>====
-
===<p style="font-size:17px; background-color:#00dd77;">153. labday 18.10.2010</p>===
+
'''Investigator: Kerstin, Anna'''
 +
<br>
-
===<p style="font-size:17px; background-color:#00dd77;">154. labday 19.10.2010</p>===
+
'''Plasmids:'''
 +
*P888: pSB1C3_001_VP3, c = 300,8 ng/µl
 +
*P890: pSB1C3_001_VP2, c = 298,3 ng/µl
 +
*P898: pSB1C3_001_VP1, c = 272,7 ng/µl
 +
*P727: pSB1C3_001_CMV, c =  225,5 ng/µl
 +
<br>
 +
'''Digestion:'''
 +
{| border="1"
 +
| <b>components</b>  || align="right" |<b>VP3</b> || align="right" |<b>VP2</b> || align="right" |<b>VP1</b>|| align="right" |<b>CMV</b>
 +
|-
 +
| DNA  ||  align="right" |4 ||  align="right" |4||  align="right" |4||  align="right" |8
 +
|-
 +
| BSA (10x) ||  align="right" |2 ||  align="right" |2||  align="right" |2||  align="right" |2
 +
|-
 +
| Buffer 4 (10x)||  align="right" |2 ||  align="right" |2 ||  align="right" |2||  align="right" |2
 +
|-
 +
|Enzyme EcoI||  align="right" |1||  align="right" |1 ||  align="right" |1||  align="right" |1
 +
|-
 +
|Enzyme XbaI||  align="right" |1||  align="right" |1 ||  align="right" |1 ||  align="right" |-
 +
|-
 +
|Enzyme SpeI||  align="right" |-||  align="right" |- ||  align="right" |-||  align="right" |1
 +
|-
 +
|H2O||  align="right" |10||  align="right" |10 ||  align="right" |10  ||  align="right" |10
 +
|-
 +
|'''Total '''||  align="right" | 20||  align="right" | 20||  align="right" | 20
 +
|}
 +
<br>
 +
*Digestion: 2h @ 37°C
 +
<br>
 +
 
 +
'''Gel:'''
 +
*1% agarose gel, 1 µl Gelred, run for 45 min
 +
 
 +
[[Image:Freiburg10_Cloning of CMV into pSB1C3_001_VP1-3.jpg|400px]]
 +
<br>
 +
 
 +
'''Gel extraction'''
 +
 
 +
{| border="1"
 +
|sample name || align="right" |<b>VP3</b>|| align="right" |<b>VP2</b>|| align="right" |<b>VP1</b>|| align="right" |<b>CMV</b>
 +
|-
 +
|nanodrop concentrations || align="right" |44,36|| align="right" |32,33|| align="right" |22,73|| align="right" |18,5
 +
|-
 +
|expected fragment size|| align="right" |4100|| align="right" |4000|| align="right" |3700|| align="right" |650
 +
|-
 +
|}
 +
 
 +
<br>
 +
'''Ligation:'''
 +
{| border="1"
 +
|ligation name || align="right" |<b>VP3 + CMV</b>|| align="right" |<b>VP2 + CMV</b>|| align="right" |<b>VP1 + CMV</b>
 +
|-
 +
|volume of vector || align="right" |5,7|| align="right" |4,3|| align="right" |5
 +
|-
 +
|volume of insert|| align="right" |3,3|| align="right" |3,7|| align="right" |3
 +
|-
 +
|T4 ligase buffer (10x)|| align="right" |1|| align="right" |1|| align="right" |1
 +
|-
 +
|T4 ligase || align="right" |1|| align="right" |1|| align="right" |1
 +
|-
 +
|}
 +
*Ligation @ RT for 30 min
 +
 
 +
'''Trafo:'''
 +
<br>
 +
 
 +
Was done following the standard protocol using BL21 cells.
 +
 
 +
 
 +
<br>
===<p style="font-size:17px; background-color:#00dd77;">155. labday 20.10.2010</p>===
===<p style="font-size:17px; background-color:#00dd77;">155. labday 20.10.2010</p>===
 +
 +
====<p style="font-size:15px; background-color:#66bbff;">Midi-Prep</p>====
 +
 +
'''Investigator: Chris W. <br>'''
 +
<p style="font-size:13px; color:#003399;"> Midi-Prep of:</p><br/>
 +
pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 1 =P903 =B702<br/>
 +
pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 1 =P904 =B712<br/>
 +
 +
 +
 +
 +
<br/>
 +
The Midi-Preps were performed according to the standard protocol yielding the following concentrations:
 +
 +
{| border="1"
 +
| plasmid-no. || align="right" |P903|| align="right" |P904
 +
|-
 +
| concentration (ng/µl)|| align="right" |832,63 || align="right" |1174,49
 +
|}
 +
<br>
 +
<br/>
 +
 +
===<p style="font-size:17px; background-color:#00dd77;">156. labday 21.10.2010</p>===
 +
====<p style="font-size:15px; background-color:#66bbff;">ÄKTA Chromatography and Ultrafiltration of virus particles </p>====
 +
 +
'''Investigator: Hanna <br>'''
 +
<b>ÄKTA chromatography</b> with VP1up_NLS_mVenus_VP2/3 containing virus particles was conducted. Fraction 5 - 10 delivered highest protein concentrations. <br/>
 +
{| border="1"
 +
|<b>Sample</b> || align="right" |<b>A(260 nm)</b> || align="right" |<b>A(280 nm)</b>|| align="right" |<b>A(515 nm) (YFP)</b>
 +
|-
 +
| 5  ||  align="right" |0.032 ||  align="right" |0.027||  align="right" |0.003
 +
|-
 +
| 6 ||  align="right" |0.019 ||  align="right" |0.019||  align="right" |0.003
 +
|-
 +
| 7||  align="right" |0.075 ||  align="right" |0.09 ||  align="right" |0.01
 +
|-
 +
|8||  align="right" |0.054||  align="right" |0.075 ||  align="right" |0.007
 +
|-
 +
|10||  align="right" |-0.008||  align="right" |-0.008 ||  align="right" |0.005
 +
|}
 +
 +
<br/>
 +
A further attempt was conducted which included digestion with Benzonase (1 hour) prior to ÄKTA chromatography. Following protein concentrations were obtained: <br/>
 +
{| border="1"
 +
|<b>Sample</b> || align="right" |<b>A(260 nm)</b> || align="right" |<b>A(280 nm)</b>|| align="right" |<b>A(515 nm) (YFP)</b>
 +
|-
 +
| 5  ||  align="right" |0.005||  align="right" |0.007||  align="right" |0.003
 +
|-
 +
| 6 ||  align="right" |0.047||  align="right" |0.041||  align="right" |0.006
 +
|-
 +
| 7||  align="right" |0.151||  align="right" |0.153||  align="right" |0.01
 +
|-
 +
|8||  align="right" |0.172||  align="right" |0.2||  align="right" |0.009
 +
|-
 +
|9||  align="right" |0.098||  align="right" |0.128||  align="right" |0.009
 +
|-
 +
|10||  align="right" |0.053||  align="right" |0.074||  align="right" |0.005
 +
|}
 +
<br/>
 +
<br/>
 +
<b>Ultrafiltration</b> <br/>
 +
Ultrafiltration of CFP_MiddleLinker_VP2/3 containing virus particles and 587-BAP virus particles were concentrated via Vivaspin-Ultrafiltration: <br/>
 +
*20 mL virus containing cell culture supernatant was added to GE Vivaspin 20 filter and centrifuged with 4000 g at 15°C until 750 - 1000 µL was left-
 +
* 5 mL Bis-Trus buffer (pH 6) was added and centrifuged again with 4000 g at 15°C (washing).
 +
* This step was repeated 3 more times.
 +
* Membrane was carefully resuspended and cleared. Suspension was transfered to low-binding eppi and centrifuged with 10000 g for 10 minutes at 15°C.
 +
* Supernatant was transfered to new low-binding eppi and again centrifuged with 10000 g for 10 minutes at 15°C.
 +
* Supernatant was transfered to new low-binding eppi and stored at 4°C over night. <b>To do:</b> ÄKTA chromatography. <br/>
 +
 +
 +
====<p style="font-size:15px; background-color:#66bbff;">MTT Assay: Testing Superconstructs </p>====
 +
'''Investigator: Anissa, Kerstin <br>'''
 +
 +
[[Image:Freiburg10 Transductionplan1 21.10.2010.jpg|700px]]
 +
[[Image:Freiburg10 Transductionplan2 21.10.2010.jpg|700px]]
 +
[[Image:Freiburg10 Transductionplan3 21.10.2010.jpg|700px]]
 +
[[Image:Freiburg10 Transductionplan4 21.10.2010.jpg|700px]]
 +
[[Image:Freiburg10 Transductionplan5 21.10.2010.jpg|700px]]
 +
 +
===<p style="font-size:17px; background-color:#00dd77;">157. labday 22.10.2010</p>===
 +
====<p style="font-size:15px; background-color:#66bbff;">SDS PAGE and Coomassie staining</p>====
 +
 +
'''Investigator: Hanna <br>'''
 +
<br/>
 +
Prior to performing Western Blot we decided to investigate running behaviour of different samples. <br/>
 +
* 1. Cell debris (control)
 +
* 2. Cell debris containing virus particles
 +
* 3. Concentrated virus stock (containing CFP_MiddleLinker_VP2/3)
 +
* 4. ÄKTA purified virus stock: Fraction 6
 +
* 5. ÄKTA purified virus stock: Fraction 7
 +
* 6. Benzonase treated, ÄKTA purified virus stock: Fraction 7
 +
* 7. Benzonase treated, ÄKTA purified virus stock: Fraction 8
 +
 +
<br/>
 +
5 µL Laemmli buffer was added to 20 µL sample. Samples were incubated at 95°C for 8 minutes and loaded onto a SDS gel (10 %). SDS PAGE was performed at 90 V (collection gel) resp. 120 V (separation gel). <br/>
 +
Gel was put into Coomassie dye, heated for 30 seconds in microwave and incubated for 1 hours shaking. <br/>
 +
Gel was decolorized in acetic acid (20%). <br/>
 +
Loading plan:
 +
{| border="1"
 +
|<b>Marker</b> || align="right" |<b>Concentrated Stock</b> || align="right" |<b>ÄKTA 6</b>|| align="right" |<b>ÄKTA 7</b>|| align="right"|<b>Benzonase/ÄKTA 7</b>|| align="right" |<b>Benzonase/ÄKTA 8</b>|| align="right"|<b> - - - </b>|| align="right" |<b> - - - </b>|| align="right"|<b>Cell debris</b>|| align="right" |<b>Cell debris + Virus</b>
 +
|}
 +
<br/>
 +
[[Image:Freiburg10 SDSVersuch2.jpg|500px|center]]
 +
<br/>
 +
Gel picture shows that concentration works :) <br/>
 +
In addition to that one can see that the BSA bands disappear after ÄKTA chromatography. <br/>
 +
<b>Next step:</b> Western Blot of ÄKTA purified virus stocks. <br/>
 +
 +
===<p style="font-size:17px; background-color:#00dd77;">158. labday 23.10.2010</p>===
 +
 +
===<p style="font-size:17px; background-color:#00dd77;">159. labday 24.10.2010</p>===
 +
 +
====<p style="font-size:15px; background-color:#66bbff;">Midi-Prep</p>====
 +
 +
'''Investigator: Chris W. <br>'''
 +
<p style="font-size:13px; color:#003399;"> Midi-Prep of:</p><br/>
 +
pSB1C3_001_RC_IRCK_VP2-ko_HSPG-ko_P5tataless cl1 =P966 =B523<br/>
 +
 +
 +
 +
 +
 +
<br/>
 +
The Midi-Prep were performed according to the standard protocol yielding the following concentration:
 +
 +
{| border="1"
 +
| plasmid-no. || align="right" |P966
 +
|-
 +
| concentration (ng/µl)|| align="right" |310,69
 +
|}
 +
<br>
 +
<br/>
 +
 +
===<p style="font-size:17px; background-color:#00dd77;">160. labday 25.10.2010</p>===
 +
 +
===<p style="font-size:17px; background-color:#00dd77;">161. labday 26.10.2010</p>===
 +
 +
===<p style="font-size:17px; background-color:#00dd77;">162. labday 27.10.2010</p>===
 +
 +
===<p style="font-size:17px; background-color:#00dd77;">163. labday 28.10.2010</p>===
 +
 +
===<p style="font-size:17px; background-color:#00dd77;">164. labday 29.10.2010</p>===
 +
 +
===<p style="font-size:17px; background-color:#00dd77;">165. labday 30.10.2010</p>===
 +
 +
===<p style="font-size:17px; background-color:#00dd77;">166. labday 31.10.2010</p>===
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<center>[https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October3 '''=> Go to Labjournal October part 3 (labday 156 - 166 )''']</center><br>
+
<center>[https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November '''=> Go to Labjournal November (labday 167 - 170 )''']</center><br>
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Latest revision as of 21:33, 27 October 2010

=> Back to Notebook overview



Contents

150. labday 15.10.2010

Cloning CFP (from P666: PSB1C3_CFP) into pSB1C3_leftITR_CMV_beta-globin (P729)

Investigator Patrick
Digestions, 2 h 10 minutes, 37 °C:

  • P666: 5 µl DNA, 2 µl BSA, 2 µl Buffer 4 (10x), 1 µl Xba, 1 µl PstI, 9 µl H2O
  • P729: 4 µl DNA, 2 µl BSA, 2 µl Buffer 4 (10x), 1 µl SpeI, 1 µl PstI, 10 µl H2O

Expected results for the 1% agarose gel:

  • P666: about 2100 and 750 bp
  • P729: about 3300 and 20 bp


Freiburg10 1015pat fertig.jpg



The gelextraction ...

P728: 11,8 ng/µl
P822: 34,6 ng/µl

... and following ligation (2,5 µl Insert, 5,5 µl vector, 1 µl T4 DNA Ligase, 1 µl T4 DNA Ligase Buffer (10x), 40 minutes, RT) were performed according to the standard protocol. After the transformation (with XL1B) the cells were plated and put into the 37°C room. Two additional transformations were performed with ligations from Volker labeled: "ligation viral brick 453 empty" & "viral brick 587 empty".

The following day the plates were checked for clones. Unfortunately there grew no clones on these two plates contrary to my plate with a a lot of clones.

Midi-Prep

Investigator: Chris W.

Midi-Prep of:


pSB1C3_001_RC_IRCK_P5tataless clone 1 =P866 =B516
pSB1C3_001_CMV_VP123_587-KO_Z34C_spacer clone2 =P867 =B526
pSB1C3_001_CMV_VP123_587-KO_Z34C clone2 =P868 =B529
pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_BAP clone 1 =P869 =B680
pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_6xHis clone 1 =P870 =B200



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

plasmid-no. P866P867P868P869P870
concentration (ng/µl)899,80 954,46 406,971642,761585,12



mini prep of several constructs

Investigator: Kira

c(rep52_1)=299,04 ng/ul
c(rep52_2)=290,07 ng/ul
c(rep78_1)=142,32 ng/ul
c(rep78_2)=175,36 ng/ul

Cell culture

Investigator: Kira

The cells are still alive. Medium was exchanged.--> RNA will be harvested tomorrow

Mini-Prep and test digestion of pSB1C3_CD_SDM-PstI_hGH_rITR

Investigator: Stefan

Glycerol stocks were prepared:

  • B694 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 1
  • B695 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 2

Mini-Prep was performed according to standard protocol:

  • P875 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 1 c = 73,1 ng/µl
  • P876 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 2 c = 78,3 ng/µl

Test digestion:

Components P875 + P876 / µl
DNA 4
Buffer 4 1
BSA (10x) 1
XbaI 0,3
AgeI 0,3
H2O 3,4
Total volume 10

Gel:
0,5g agarose, 50 ml TAE (1%), 3 µl GELRED, 115 Volt, running time ~50 minutes

Freiburg10 td151010.jpg

Comment: Test digestion looks allright, cloning will be continued using P876.

151. labday 16.10.2010

Biobrick assembly: pSB1C3_lITR_CMV_ß-globin_CD_hGH_rITR and pSB1C3_lITR_phTERT_ß-globin_CD_hGH_rITR

Investigator: Achim

Plasmids:

  • P729: pSB1C3_lITR_CMV_ß-Globin
    • c= 243.4 ng/µl
  • P730: pSB1C3_lITR_pHTERT_ß-Globin
    • c= 81.1 ng/µl
  • P876: pSB1C3_CD_SDM-PstI_hGH_rITR
    • c= 78.3 ng/µl

Digestion:

components I1 (P729) I2 (P730) V (P876)
DNA 6 1414
BSA (10x) 2 22
Buffer 4 (10x)2 2 2
Enzyme EcoI11 1
Enzyme XbaI-- 1
Enzyme SpeI11 -
H2O8- -
Total 20 20 20

Digestion: 2h, 37°C

Prep. gel:

  • 0,8%, run for 45 min
Expected Bands: I1: 1335, I2: 1138, V: 4000
  • Corresponding bands were cut out

Gel ex.

  • Nanodrop concentrations:
    • I1: 37.54 ng/µl
    • I2: 25.38 ng/µl
    • V: 26.47 ng/µl

Ligation:

ligation name I1 + VI2 + V
volume of vector 4.694.23
volume of insert3.313.77
T4 ligase buffer (10x)11
T4 ligase 11
  • Ligation @ RT for 40 min

Trafo:

  • Done by Kira



Mini-prep of mutual pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI

Investigator Patrick

Yielded concentrations & given numbers:

  • pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 1: 208,4 ng/µl , P877 / B696
  • pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 2: 251,6 ng/µl , P878 / B696
  • pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 3: 212,6 ng/µl , P889 / B696



Test-digestion: 0,5 µl SpeI, 0,5 µl PstI, 3 µl DNA, 1 µl Buffer 4, 1 µl BSA, 4 µl H2O, 40 minutes, 37°C
Expected results: fragments with about 650 and 4900 bp

Freiburg10 1016pat test.jpg


Obviously, this test digestion has to be repeated.

Preparations for tomorrow:

  • Mini-prep of 3 mutual pSB1C3_leftITR_CMV_beta-globin_CFP clones (have to be picked from the plate)
  • Midi-prep of pHelper
  • Midi-prep of B689:pSB1C3_lITR_CMV_betaglobin_mGMK_TK30_SDM-PstI_hGH_rITR clone 2

Biobrick assembly of Rep78 and Rep52

Investigator: Kira
Comment: After replacing the mutated Rep parts by the ordered Rep parts, PCR amplification has to be done in order to produce a biobrick. PCR program:

c(Rep52)=299 ng/ul
c(Rep78)=175 ng/ul

Rep52: praefix 094 & suffix 097
Rep78: praefix 093 & suffix 097


components volume in µl
5x Phusion HF buffer 10
10 mM dNTP mix 1
primer_for (1:10 dilution) 2,5
primer_rev (1:10 dilution) 2,5
DNA template (1:100) 0,5
DMSO 0,5
Phusion polymerase 0,5
H2O 32,5
Total volume (e.g. 50 µl) 50


CyclesTemperatureTime
98°C30 sec
10x98°C15 sec
63°C25 sec
72°C32 sec
20x98°C15 sec
66°C25 sec
72°C32 sec
1x72°C5 min
Hold 4°C


1% agarose gel
Freiburg10 Rep78&Rep52 PCR.jpg

Digestion of plasmid backbone:

pSB1C3_001 is used as backbone

Components <b>vector Volume/µL
DNA 3,5 µl
BSA (10x) 2 µl
Buffer no. 4 (10x) 2,0 µl
Enzyme 1 EcoRI-HF 0,5 µl
Enzyme 2 SpeI 1,0 µl
H2O 15 µl
Total volume 25


incubation @ 37 C for approx. 2 h

1% agarose gel

Freiburg10 digestion pSB1C31 16.10.jpg

Digestion of PCR product:

Components PCR product Volume/µL
DNA 35,0 µl
BSA (100x) 0,45 µl
Buffer no. 4 4,5 µl
Enzyme 1 EcoRI-HF 1,5 µl
Enzyme 2 SpeI 2,0 µl
H2O 1,5 µl
Total volume 45


incubation @ 37 C for approx. 2 h

T4 ligation for 40 min
Transformation according to the standard protocol

RNA harvesting

Investigator: Kira
After transfection, the cells were incubated for 48 hours. Today, the cells will be harvested and RNA extracted, in order to perform RT-PCR and an additional PCR for evaluation of promoter activity.

The transfected cells were trypsinised and centrifuged for 2 min. The supernatant was discarded and pellet washed 2x with PBS. RNeasy Kit [Qiagen] was used for RNA extraction according to the manufacturer protocol.

c(CMV)= 335,69 ng/ul
c(P40)= 857,92 ng/ul
c(AAV_RC)= 760,21 ng/ul

152. labday 17.10.2010

Test digestion of pSB1C3_lITR_CMV_ß-globin_CFP

Investigator: Anna

Vector name:
pSB1C3_lITR_CMV_betaglobin_CFP_cl1 (P880): c = 452,67 ng/µl
pSB1C3_lITR_CMV_betaglobin_CFP_cl2 (P881): c = 288,88 ng/µl
pSB1C3_lITR_CMV_betaglobin_CFP_cl3 (P882): c = 288,36 ng/µl

Test Digestion:

components volume P880 - P882 /µl volume P434 /µl
DNA 2 2
BSA (10x) 1 1
Buffer 4 (10x)1 1
Enzyme NgoMIV0,30,3
Enzyme AgeI0,30,3
H2O5,45,4
Total volume 10 10


Gel:
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt



Freiburg10 Test digestion of pSB1C3 lITR CMV ß-globin CFP.jpg

Cloning of hGH_rITR into pSB1C3_lITR_CMV_betaglobin_CFP

Investigator: Stefan

Cloning of our last GOI!


Vector name:
pSB1C3_lITR_CMV_betaglobin_CFP cl 1-3 (P880-P882)

Insert name:
pSB1C3_hGH_rITR (P728)

Digestion:


components volume P880 - P882 /µl volume P728 /µl
DNA 3 8
BSA (10x) 2 2
Buffer 4 (10x)2 2
Enzyme PstI11
Enzyme XbaI-1
Enzyme SpeI1-
H2O114
Total volume (e.g. 15,20,25,30 µl) 20 20


Gel:
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt



Freiburg10 171010.jpg

Test digestion of all constructs looked alright, therefore, cloning was continued using P881 only.



Gel extraction:
Was performed according to protocol.


T4 Ligation:

ligation name 728 + 881
volume of vector 2,68
volume of insert5,32
T4 ligase buffer (10x)1
T4 ligase 1


Transformation:
Transformation was performed according to standard protocol using BL21 cells.

RT-PCR

Investigator: Kira
For further experiments, RNA has to be translated into cDNA. The PCR was performed according to the manufacturer protocol.

153. labday 18.10.2010

quantitative real-time PCR for detection of virus titer

Investigator: Achim

  • qPCR of harvested virus particles to determine the virus titers of our different constructs
  • Total number of samples: 58

Test-digestion of pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 1, 2 and 3 (P877, 878 and 879)

Investigator Patrick

Check the plasmid for leftITR: 0,5 µl EcoRI, 1 µl BstEII, 7 µl DNA, 2 µl Buffer 4, 2 µl BSA, 7,5 µl H2O, 45 minutes 37°C, 45 minutes 60°C

Check the plasmid for hGH_rITR and ... : 0,5 µl AgeI, 0,5 µl PstI, 3 µl DNA, 1 µl BSA, 1 µl Buffer 4, 4 µl H2O, 70 minutes 37°C

Expected results:

  • leftITR: about 190 bp
  • hGH_rITR: about 670 bp


Unfortunately the digestions had to be reapeated because i didnt switch on the current so the samples and especially the 1kb GeneRuler marker diffused.

The second run: see above
Freiburg10 1019pat test1bfertig.jpg
PstI or BstEII seems to work not properly

MTT Assay

Investigator Kerstin, Anissa

Freiburg10 transductionplan 18.10..jpg

Freiburg10 transductionplan1 18.10..jpg
Freiburg10 transductionplan2 18.10..jpg

Results:

Freiburg10 Results of MTTAssay 18 10 10 2day 1pic.jpg
Freiburg10 Results of MTTAssay 18 10 10 2day 3pic.jpg
Freiburg10 Results of MTTAssay 18 10 10 2day 2pic.jpg

Mini-Prep and test digestion of several constructs

Investigator: Jessica

Glycerol stocks were prepared:

  • B702 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 1
  • B703 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 2
  • B704 = pSB1C3_001_VP3 clone 1
  • B705 = pSB1C3_001_VP3 clone 2
  • B706 = pSB1C3_001_VP2 clone 1
  • B707 = pSB1C3_001_VP2 clone 2
  • B708 = pSB1C3_001_Rep78 clone 1
  • B709 = pSB1C3_001_Rep78 clone 2
  • B710 = pSB1C3_001_Rep52 clone 1
  • B711 = pSB1C3_001_Rep52 clone 2
  • B712 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 1
  • B713 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 2
  • B714 = pSB1C3_001_VP1 clone 1
  • B715 = pSB1C3_001_VP1 clone 2

Mini-Prep was performed according to standard protocol:

  • P886 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 1 c= 232,2ng/µl
  • P887 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 2 c= 186,2ng/µl
  • P888 = pSB1C3_001_VP3 clone 1 c= 300,8ng/µl
  • P889 = pSB1C3_001_VP3 clone 2 c= 284,4ng/µl
  • P890 = pSB1C3_001_VP2 clone 1 c= 298,3ng/µl
  • P891 = pSB1C3_001_VP2 clone 2 c= 299,9ng/µl
  • P892 = pSB1C3_001_Rep78 clone 1 c= 143,9ng/µl
  • P893 = pSB1C3_001_Rep78 clone 2 c= 163,4ng/µl
  • P894 = pSB1C3_001_Rep52 clone 1 c= 166,6ng/µl
  • P895 = pSB1C3_001_Rep52 clone 2 c= 181,6ng/µl
  • P896 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 1 c= 250,5ng/µl
  • P897 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 2 c= 173,4ng/µl
  • P898 = pSB1C3_001_VP1 clone 1 c= 272,7ng/µl
  • P899 = pSB1C3_001_VP1 clone 2 c= 294,8ng/µl
  • P900 = pSB1C3_hGH_rITR (from B160) c= 136,7ng/µl

Test digestion:

Components P886,887,892,893,894,895,896,897 / µl P888,889,890,891898,899 / µl
DNA 1,5 1,5
Buffer (4) 1 (2) 1
BSA (10x) 1 1
NgoMIV 0,4 -
XbaI 0,4 -
PstI - 0,6
XcmI - 0,4
H2O 4,5 4,5
Total volume 10 10

Gel:
1,0g agarose, 100 ml TAE (1%), 6 µl GELRED, Volt, running time minutes

Freiburg10 test digestion 886-899.jpg

Comment: Rep 52/78 will be checked,pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR and pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR will be sequenced

PCR of mGMK and SR39

Investigator: Anna


Plasmids:
pSB1C3_mGMK_TK30_SDM-PstI clone 2(P804)
pSB1C3_mGMK_sr39 clone 1(P860)

Oligos:
O193: pTK30_for
O81: pmgmk_tk30_suffix_RFC25_rev


PCR Mix:

Components Volume /µl
Phusion Buffer 10
dNTP 1
Primer_for2,5
Primer_rev2,5
DNA template1
H2O32,5
Total volume 50


PCR Program:

CyclesTemperatureTime
98°C60 sec
98°C15 sec
8x52°C25 sec
72°C25 sec
98°C15 sec
17x67°C25 sec
72°C25 sec
1x72°C5 min
Hold 4°C


Gel:
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt



[[Image:|550px|]]

154. labday 19.10.2010

Midi-Prep

Investigator: Chris W.

Midi-Prep of:


pSB1C3_lITR_CMV_betaglobin_mVenus_hGH_rITR clone1 =P901 =B200
pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 2 =P902 =B697




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

plasmid-no. P901P902
concentration (ng/µl)1563,63 1348,26



Continuation of PCR of mGMK and SR39

Investigator: Jessica

  • vector (P320) and PCR product was digested
Components P320 / µl PCR product P804 and P860 / µl
DNA 1,5 20
Buffer 2 3
BSA (10x) 2 3
AgeI 1 1,5
XbaI 1 1,5
H2O 14,5 1
Total volume 20 30
Freiburg10 pcr mGMK and SR39.jpg



Ligation

  • P320 c= 5,08 ng/µl
  • P804 c= 11,73 ng/µl
  • P860 c= 26,57 ng/µl


  • P320 + P804: 4,93µl : 3,07µl
  • P320 + P860: 6,28µl : 1,72µl


Transformation with BL21 and Cm

Cloning of CMV into pSB1C3_001_VP1, pSB1C3_001_VP2 and pSB1C3_001_VP3

Investigator: Kerstin, Anna

Plasmids:

  • P888: pSB1C3_001_VP3, c = 300,8 ng/µl
  • P890: pSB1C3_001_VP2, c = 298,3 ng/µl
  • P898: pSB1C3_001_VP1, c = 272,7 ng/µl
  • P727: pSB1C3_001_CMV, c = 225,5 ng/µl


Digestion:

components VP3 VP2 VP1CMV
DNA 4 448
BSA (10x) 2 222
Buffer 4 (10x)2 2 22
Enzyme EcoI11 11
Enzyme XbaI11 1 -
Enzyme SpeI-- -1
H2O1010 10 10
Total 20 20 20


  • Digestion: 2h @ 37°C


Gel:

  • 1% agarose gel, 1 µl Gelred, run for 45 min

Freiburg10 Cloning of CMV into pSB1C3 001 VP1-3.jpg

Gel extraction

sample name VP3VP2VP1CMV
nanodrop concentrations 44,3632,3322,7318,5
expected fragment size410040003700650


Ligation:

ligation name VP3 + CMVVP2 + CMVVP1 + CMV
volume of vector 5,74,35
volume of insert3,33,73
T4 ligase buffer (10x)111
T4 ligase 111
  • Ligation @ RT for 30 min

Trafo:

Was done following the standard protocol using BL21 cells.



155. labday 20.10.2010

Midi-Prep

Investigator: Chris W.

Midi-Prep of:


pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 1 =P903 =B702
pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 1 =P904 =B712




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

plasmid-no. P903P904
concentration (ng/µl)832,63 1174,49



156. labday 21.10.2010

ÄKTA Chromatography and Ultrafiltration of virus particles

Investigator: Hanna
ÄKTA chromatography with VP1up_NLS_mVenus_VP2/3 containing virus particles was conducted. Fraction 5 - 10 delivered highest protein concentrations.

Sample A(260 nm) A(280 nm)A(515 nm) (YFP)
5 0.032 0.0270.003
6 0.019 0.0190.003
70.075 0.09 0.01
80.0540.075 0.007
10-0.008-0.008 0.005


A further attempt was conducted which included digestion with Benzonase (1 hour) prior to ÄKTA chromatography. Following protein concentrations were obtained:

Sample A(260 nm) A(280 nm)A(515 nm) (YFP)
5 0.0050.0070.003
6 0.0470.0410.006
70.1510.1530.01
80.1720.20.009
90.0980.1280.009
100.0530.0740.005



Ultrafiltration
Ultrafiltration of CFP_MiddleLinker_VP2/3 containing virus particles and 587-BAP virus particles were concentrated via Vivaspin-Ultrafiltration:

  • 20 mL virus containing cell culture supernatant was added to GE Vivaspin 20 filter and centrifuged with 4000 g at 15°C until 750 - 1000 µL was left-
  • 5 mL Bis-Trus buffer (pH 6) was added and centrifuged again with 4000 g at 15°C (washing).
  • This step was repeated 3 more times.
  • Membrane was carefully resuspended and cleared. Suspension was transfered to low-binding eppi and centrifuged with 10000 g for 10 minutes at 15°C.
  • Supernatant was transfered to new low-binding eppi and again centrifuged with 10000 g for 10 minutes at 15°C.
  • Supernatant was transfered to new low-binding eppi and stored at 4°C over night. To do: ÄKTA chromatography.


MTT Assay: Testing Superconstructs

Investigator: Anissa, Kerstin

Freiburg10 Transductionplan1 21.10.2010.jpg Freiburg10 Transductionplan2 21.10.2010.jpg Freiburg10 Transductionplan3 21.10.2010.jpg Freiburg10 Transductionplan4 21.10.2010.jpg Freiburg10 Transductionplan5 21.10.2010.jpg

157. labday 22.10.2010

SDS PAGE and Coomassie staining

Investigator: Hanna

Prior to performing Western Blot we decided to investigate running behaviour of different samples.

  • 1. Cell debris (control)
  • 2. Cell debris containing virus particles
  • 3. Concentrated virus stock (containing CFP_MiddleLinker_VP2/3)
  • 4. ÄKTA purified virus stock: Fraction 6
  • 5. ÄKTA purified virus stock: Fraction 7
  • 6. Benzonase treated, ÄKTA purified virus stock: Fraction 7
  • 7. Benzonase treated, ÄKTA purified virus stock: Fraction 8


5 µL Laemmli buffer was added to 20 µL sample. Samples were incubated at 95°C for 8 minutes and loaded onto a SDS gel (10 %). SDS PAGE was performed at 90 V (collection gel) resp. 120 V (separation gel).
Gel was put into Coomassie dye, heated for 30 seconds in microwave and incubated for 1 hours shaking.
Gel was decolorized in acetic acid (20%).
Loading plan:

Marker Concentrated Stock ÄKTA 6ÄKTA 7Benzonase/ÄKTA 7Benzonase/ÄKTA 8 - - - - - - Cell debrisCell debris + Virus


Freiburg10 SDSVersuch2.jpg


Gel picture shows that concentration works :)
In addition to that one can see that the BSA bands disappear after ÄKTA chromatography.
Next step: Western Blot of ÄKTA purified virus stocks.

158. labday 23.10.2010

159. labday 24.10.2010

Midi-Prep

Investigator: Chris W.

Midi-Prep of:


pSB1C3_001_RC_IRCK_VP2-ko_HSPG-ko_P5tataless cl1 =P966 =B523




The Midi-Prep were performed according to the standard protocol yielding the following concentration:

plasmid-no. P966
concentration (ng/µl)310,69



160. labday 25.10.2010

161. labday 26.10.2010

162. labday 27.10.2010

163. labday 28.10.2010

164. labday 29.10.2010

165. labday 30.10.2010

166. labday 31.10.2010

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