Team:LMU-Munich/Notebook/Apoptosis

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__NOTOC__
__NOTOC__
{{:Team:LMU-Munich/Templates/Page Header}}
{{:Team:LMU-Munich/Templates/Page Header}}
-
=Apoptosis Notebook=
+
==<font color="#9933CC">'''ApoControl Notebook'''</font>==
-
=='''Contents'''==
+
-
{| class="wikitable centered" border="2" rules="rows" width="100%" style="border-color:white;"
+
== '''What we did''' ==
 +
<b>Short description of our work, our results and our supporters</b>
 +
 
 +
 
 +
The creation of certain constructs was necessary for our two systems for cell selection by means of apoptosis: “Cut’N’Survive” and “Jump-Or-Die”. We searched for sources of the DNA sequences we needed and found several supporters which are listed below.
 +
 
 +
Most genes and promoters were amplificated via PCR with overhang-primers with the BioBrick prefix or suffix. If the sequence contained a EcoR1-, Pst1-, Xba1-, Spe1- or Not1- restriction site, we used mutagenesis primers and fusioned both DNA parts by fusion PCR. All PCRs worked out, even the fusion PCRs.
 +
 
 +
The length of the PCR products were tested by agarose gel electrophoresis. We tried to sequence our PCR products, but obtained poor results and resorted to sequencing the plasmids.
 +
 
 +
In parallel, we made competent cells and multiplied ccdB (death gene)-vectors with different antibiotic resistances. All components were digested with the appropriate restriction enzymes. The samples were cleaned with a PCR clean up kit or dephosphorylated to reduce false ligations.
 +
 
 +
We ligated our constructs and several interim stages with the 3A-assembly according to our schedule. The ligations were transformed to E.coli DH5α strains and selected by antibiotics. Afterwards, some colonies were picked and we tested the insertion of the construct by colony PCR.
 +
 
 +
If the colony PCR resulted in bands of the right size, we extracted the plasmids from overnight cultures and sequenced the samples with forward and reverse BioBrick primers.
 +
 
 +
Unfortunately, not all BioBricks were cloned succesfully. However, we were able to produce 4 BioBricks, one of which represents a full construct while the other three are intermediates. The system wasn't completed on time, so we weren´t able to test them in eukarytic cell lines.
 +
 
 +
 
 +
 
 +
<b>The protocols we used are listed here: </b> [[Team:LMU-Munich/Notebook#Protocols|Protocols]]
 +
 
 +
<b>These Biobricks we submitted: </b>
 +
 
 +
*BBa_K368004: attP+eGFP+SV40PA
 +
*BBa_K368011: eGFP+SV40PA
 +
*BBa_K368016: TEVrecognition site+N-degron+SF3b155
 +
*BBa_K368019: TEV-Protease+p14*+TEVrecognition site
 +
 
 +
<b>Sources, helpers and supporters:</b>
 +
 
 +
* Prof. Dr. Angelika Böttger :
 +
** prevTRE (tet-on CMV promoter; inducible by doxycycline in special cell lines)
 +
** supported the construction ideas and would have given us the cells and mediums we would have needed
 +
** SV40PA (Polyadenylation site): gave us a vector containing it
 +
** Human Bak:  her assistant Erika Clement gave us appropriate cDNA
 +
 
 +
* Dr. Arnim Weber: submitted us a vector with human Bak
 +
* Dr. Philipe Soriano: <html>
 +
<a href="http://www.ncbi.nlm.nih.gov/pubmed/17225864?dopt=Abstract"> (Raymond CS et al: High-Efficiency FLP and PhiC31 Site-Specific Recombination in Mammalian Cells (2007))</a>
 +
</html>
 +
** Sequences of attB and attP site
 +
** PhiC31o was bought via addgene
 +
* Knop, M (Heidelberg): <html>
 +
<a href ="http://www.ncbi.nlm.nih.gov/pubmed?term=Efficient%20protein%20depletion%20by%20genetically%20controlled%20deprotection%20of%20a%20dormant%20N-degron">(Knop et al.: Efficient protein depletion by genetically controlled deprotection of a dormant N-degron (2009))</a>
 +
</html>
 +
** TEVrecognition site+N-degron+SF3b155
 +
** TEV-Protease+p14*+TEVrecognition site
 +
* Prof. Dr. Thorsten Mascher:
 +
** Helped with primer design, agarose gel electrophoresis apparatuses and trouble shooting
 +
* Prof. Dr. Kirsten Jung:
 +
** Helped with ideas and fundraising
 +
* Dr. Susanne Gebhard:
 +
** Helped with trouble-shooting and materials
 +
* Prof. Dr. Andreas Brachmann:
 +
** Sequenced our samples
 +
* Partsregistry:
 +
** eGFP (BBa_I714891)
 +
** CMV-Promoter (BBa_J52034: this part was wrong: its lacI !!!)
 +
** ccdB amp, cam, tet, kan in E.coli DH3
 +
 
 +
=='''Contents'''>==
 +
 
 +
 
 +
{|class="wikitable centered" border="2" rules="rows" width="100%" style="border-color:white;"
|-   
|-   
! Week                      !! colspan="7" |Days
! Week                      !! colspan="7" |Days
Line 97: Line 160:
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-02-2010|10-02-2010]]
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-02-2010|10-02-2010]]
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-03-2010|10-03-2010]]
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-03-2010|10-03-2010]]
 +
|-
 +
|style="text-align:center"| 40
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-04-2010|10-04-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-05-2010|10-05-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-05-2010|10-06-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-07-2010|10-07-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-08-2010|10-08-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-09-2010|10-09-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-10-2010|10-10-2010]]
 +
|-
 +
|style="text-align:center"| 41
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-11-2010|10-11-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-12-2010|10-12-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-13-2010|10-13-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-14-2010|10-14-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-15-2010|10-15-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-16-2010|10-16-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-17-2010|10-17-2010]]
 +
|-
 +
|style="text-align:center"| 42
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-18-2010|10-18-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-19-2010|10-19-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-20-2010|10-20-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-21-2010|10-21-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-22-2010|10-22-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-23-2010|10-23-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-24-2010|10-24-2010]]
 +
|-
 +
|style="text-align:center"| 43
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-25-2010|10-25-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-26-2010|10-26-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-27-2010|10-27-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-28-2010|10-28-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-29-2010|10-29-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-30-2010|10-30-2010]]
 +
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-31-2010|10-31-2010]]
|-  
|-  
|}
|}
-
 
-
== 8-02-2010 ==
 
-
 
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* Some test text here.
 
-
 
-
== 8-03-2010 ==
 
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'''Some test text in bold'''
 
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We created following tests:
 
-
* test1
 
-
* test2
 
-
* test3
 
-
 
-
- test4<br/>
 
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- test5
 
-
 
-
== 8-04-2010 ==
 
-
Example of a table
 
-
{| class="wikitable" border="1"
 
-
|-
 
-
! header 1
 
-
! header 2
 
-
! header 3
 
-
|-
 
-
| row 1, cell 1
 
-
| row 1, cell 2
 
-
| row 1, cell 3
 
-
|-
 
-
| row 2, cell 1
 
-
| row 2, cell 2
 
-
| row 2, cell 3
 
-
|}
 
-
 
-
== 8-05-2010 ==
 
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this too is a table:
 
-
{|
 
-
|H<sub>2</sub>O<sub>ddes</sub>
 
-
|10,3 µl
 
-
 
-
|-
 
-
|RE10 + Buffer H
 
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|2,0 µl
 
-
 
-
|-
 
-
|acetylated BSA   
 
-
|0,2 µl
 
-
 
-
|-
 
-
|DNA
 
-
|6,0 µl
 
-
|}
 
-
 
-
 
-
 
-
table with 3 cells
 
-
{|
 
-
|  apple    ||  banana    ||  peaches
 
-
|-
 
-
|  green    ||  yellow    ||  red
 
-
|}
 
-
 
-
== 8-06-2010 ==
 
-
text
 
-
 
-
== 8-07-2010 ==
 
-
text
 
-
 
-
== 8-08-2010 ==
 
-
test <br> test
 
-
 
-
== 8-09-2010 ==
 
-
text <font color="#FF0000">Knallroter Text</font><br>farbnummern für farbige schrift: http://html.nicole-wellinger.ch/hilfen/farbenverzeichnis.html
 
-
 
-
<font color="#009933">test grüner text</font>
 
== 8-10-2010 ==
== 8-10-2010 ==
Line 214: Line 240:
- 3 ml LB-Media + 4 µl Kanamycin
- 3 ml LB-Media + 4 µl Kanamycin
-
- Inoculated iangeimpft) with 1 colony of BBa_I714891 -> 37°C
+
- Inoculated with 1 colony of BBa_I714891 -> 37°C
Line 224: Line 250:
-
<font color="#009933">Restriction digest (Restriktionsverdau) of CMV-Promoter BBa_J52034 with EcoRI and PstI</font>
+
<font color="#009933">Restriction digestion of CMV-Promoter BBa_J52034 with EcoRI and PstI</font>
{|
{|
Line 268: Line 294:
-      10µg DNA: pDS7 (2µl), eGFP (15µl), 190-6 (10µl)
-      10µg DNA: pDS7 (2µl), eGFP (15µl), 190-6 (10µl)
-
<font color="#009933">Plate colonies for plasmid extrction</font>
+
<font color="#009933">Plate colonies for plasmid extraction</font>
- CMV (Kanamycin), eGFP (Kanamycin), pDS7 (Ampicillin), 190-6 (Ampicillin))  
- CMV (Kanamycin), eGFP (Kanamycin), pDS7 (Ampicillin), 190-6 (Ampicillin))  
Line 282: Line 308:
[[Image:GelverdauCMVpDS71906eGFP.jpg|thumb|right|Gelfoto from the EcoR1 and Pst1 Restrictiondigest of 190-6, eGFP, pDS7 and CMV]]
[[Image:GelverdauCMVpDS71906eGFP.jpg|thumb|right|Gelfoto from the EcoR1 and Pst1 Restrictiondigest of 190-6, eGFP, pDS7 and CMV]]
-
<font color="#009933">Inoculate CMV into LB medium with amicillin</font>
+
<font color="#009933">Inoculate CMV into LB medium with ampicillin</font>
- CMV (BBa_J52034) from 10.8.2010 inoculated into LB medium with ampicillin, as falsly inoculated in Kanamycin
- CMV (BBa_J52034) from 10.8.2010 inoculated into LB medium with ampicillin, as falsly inoculated in Kanamycin
Line 294: Line 320:
- expected DNA bands: 190-6 (4840bp, 1903bp), pDS7 (8027bp, 6bp), CMV (654 bp (Insert), 2079bp (Plasmid)), eGFP (720bp (Insert), 2750bp (Plasmid))
- expected DNA bands: 190-6 (4840bp, 1903bp), pDS7 (8027bp, 6bp), CMV (654 bp (Insert), 2079bp (Plasmid)), eGFP (720bp (Insert), 2750bp (Plasmid))
-
- Correct DNA bands for 190-6 (~4800bp, ~1900bp, ~6700bp (undigested plasmid)) and eGFP (~2000bp (Plasmid), ~750 bp (Insert)); CMV probably not digested (two bands; one probably normal, one supercoiled) and pDS7 not clear
+
- <font color="#CC33CC">'''Correct DNA bands for 190-6 (~4800bp, ~1900bp, ~6700bp (undigested plasmid)) and eGFP (~2000bp (Plasmid), ~750 bp (Insert))'''</font>; CMV probably not digested (two bands; one probably normal, one supercoiled) and pDS7 not clear
<font color="#009933">Restriction digest from CMV and pDS7</font>
<font color="#009933">Restriction digest from CMV and pDS7</font>
Line 310: Line 336:
- Expected DNA bands: CMV see above, pDS7 (3647bp, 3369bp, 1011bp, 6bp)
- Expected DNA bands: CMV see above, pDS7 (3647bp, 3369bp, 1011bp, 6bp)
-
- false DNA bands CMV (~1200 bp, ~2000 bp) and pDS7 (~8000bp two bands, ~1100 bp); required to isolate a new colony for these two Plasmidextractions  
+
- false DNA bands CMV (~1200 bp, ~2000 bp) and pDS7 (~8000bp two bands, ~1100 bp); required to isolate a new colony for these two Plasmidextractions
<font color="#009933">Plated CMV on Ampicllin-Agar</font>
<font color="#009933">Plated CMV on Ampicllin-Agar</font>
Line 386: Line 412:
|-
|-
|
|
-
|pcDNA3 (0,6 µg/µl)
 
|pTRERev (0,15µg/µl)
|pTRERev (0,15µg/µl)
 +
|pcDNA3 (0,6 µg/µl)
|-
|-
Line 498: Line 524:
- Agarose gel electrophoresis of the restriction digest of PhiC31o and PCR 1 and 6
- Agarose gel electrophoresis of the restriction digest of PhiC31o and PCR 1 and 6
-
- the right bands found for PhiC31o (~2900,~2400,~250)
+
- <font color="#CC33CC">'''the right bands found for PhiC31o (~2900,~2400,~250)'''</font>
-
- the right band found for PCR1 (~450)
+
- <font color="#CC33CC">'''the right band found for PCR1 (~450)'''</font>
- no band found for PCR6; new electrophoresis needed with more DNA loaded
- no band found for PCR6; new electrophoresis needed with more DNA loaded
Line 527: Line 553:
- new agarose gel electrophoresis from PCR6 with 5µl DNA instead of 3µl (image not yet shown)
- new agarose gel electrophoresis from PCR6 with 5µl DNA instead of 3µl (image not yet shown)
-
- the right band found for PCR6 (~200)
+
-<font color="#CC33CC"> '''the right band found for PCR6 (~200)'''</font>
<font color="#009933">New overnight cultures of CMV and pDS7</font>
<font color="#009933">New overnight cultures of CMV and pDS7</font>
Line 556: Line 582:
<font color="#009933">Restriction digestion</font>
<font color="#009933">Restriction digestion</font>
-
-> Protocol ([[Team:LMU-Munich/Notebook/Protocols/5_Restriction_digest|5 Restriction digest]])
+
-> Protocol ([[Team:LMU-Munich/Notebook/Protocols/5_Restriction_digest|5 Restriction digestion]])
- Restriction digestion of CMV (EcoR1 + Pst1; 10µl DNA, buffer H) and pDS7 (EcoR1 + Spe1; 5µl DNA, buffer B)
- Restriction digestion of CMV (EcoR1 + Pst1; 10µl DNA, buffer H) and pDS7 (EcoR1 + Spe1; 5µl DNA, buffer B)
Line 566: Line 592:
-> Protocol ([[Team:LMU-Munich/Notebook/Protocols/11_Agarose_gel_electrophoresis|11 Agarorse gel electrophoresis]])
-> Protocol ([[Team:LMU-Munich/Notebook/Protocols/11_Agarose_gel_electrophoresis|11 Agarorse gel electrophoresis]])
-
-> right DNA bands for pDS7 (~7000bp, ~1000bp)
+
-> <font color="#CC33CC">'''right DNA bands for pDS7 (~7000bp, ~1000bp)'''</font>
-> false DNA bands for CMV
-> false DNA bands for CMV
Line 585: Line 611:
|}
|}
-
-> the right bands for PCR2a (~300bp) and PCR2b (~700bp)
+
-><font color="#CC33CC"> '''the right bands for PCR2a (~300bp) and PCR2b (~700bp)'''</font>
- New agarose gel electrophoresis with all of the PCR product for gel extraction (150V, 30min)
- New agarose gel electrophoresis with all of the PCR product for gel extraction (150V, 30min)
Line 608: Line 634:
[[Image:GelverdauPCR3-1.jpg|400px|Gelfoto from PCR3]]
[[Image:GelverdauPCR3-1.jpg|400px|Gelfoto from PCR3]]
-
left column: marker; most right column: PCR3
+
left column: marker; rightmost column: PCR3
-> Protocol:  [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] (150V, 30min)
-> Protocol:  [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] (150V, 30min)
Line 728: Line 754:
<font color="#009933">PCR 2a gel extraction</font>
<font color="#009933">PCR 2a gel extraction</font>
-
- Quaigen kit (QuaiexII)
+
- Quiagen kit (QuiaexII)
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/14 QIAEX II gel extraction| 14 QIAEX II gel extraction]]
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/14 QIAEX II gel extraction| 14 QIAEX II gel extraction]]
Line 740: Line 766:
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
-
[[Image:24_8_10_apo3.jpg|thumb|right|Agarose gel electrophoresis of (from left to right) CMV (2ng (cut out), 10ng, 5ng template) showing the right bands for 2ng, 5ng template]]
+
[[Image:24_8_10_apo3.jpg|thumb|right|Agarose gel electrophoresis of (from left to right) PCR2b (2ng (cut out), 10ng, 5ng template) showing the right bands for 2ng, 5ng template]]
-
- expected bands: right bands with 2ng and 5ng template (~700bp), no band with 10ng template
+
- expected bands:<font color="#CC33CC"> '''right bands with 2ng and 5ng template (~700bp)'''</font>, no band with 10ng template
<font color="#009933">CMV plasmid extraction</font>
<font color="#009933">CMV plasmid extraction</font>
Line 755: Line 781:
* 80 ng/µl A260/A280= 1.600
* 80 ng/µl A260/A280= 1.600
-
<font color="#009933">CMV restriction digest</font>
+
<font color="#009933">CMV restriction digestion</font>
-
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/5 Restriction digest|5 Restriction digest]]
+
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/5 Restriction digest|5 Restriction digestion]]
- CMV restriction digest: EcoRI, PstI, buffer H
- CMV restriction digest: EcoRI, PstI, buffer H
Line 842: Line 868:
<font color="#009933">Plate ccdB amp, cam, tet</font>
<font color="#009933">Plate ccdB amp, cam, tet</font>
-
- plate ccdB with ampicilline, chloramphenicol, tetracycline resistence on LB agar with appropiate antibiotic.
+
- plate ccdB with ampicilline, chloramphenicol, tetracycline resistance on LB agar with appropiate antibiotic.
<font color="#009933">Overnight culture of ccdB kan</font>
<font color="#009933">Overnight culture of ccdB kan</font>
-
- Overnight culture of ccdB with kanamycine resistence in LB medium with kanamycine
+
- Overnight culture of ccdB with kanamycine resistance in LB medium with kanamycine
<font color="#009933">PCR 7a, 7b, 9, 10</font>
<font color="#009933">PCR 7a, 7b, 9, 10</font>
Line 1,048: Line 1,074:
|-
|-
-
|7b
+
|<font color="#CC33CC">'''7b'''</font>
-
|402bp
+
|<font color="#CC33CC">'''402bp'''</font>
-
|right band (~400bp)+ false band (~150bp)
+
|<font color="#CC33CC">'''right band (~400bp)'''</font>+ false band (~150bp)
|-
|-
Line 1,058: Line 1,084:
|-
|-
-
|10
+
|<font color="#CC33CC">'''10'''</font>
-
|1888bp
+
|<font color="#CC33CC">'''1888bp'''</font>
-
|right band (~1900bp)+false band (~500bp)
+
|<font color="#CC33CC">'''right band (~1900bp)'''</font>+false band (~500bp)
|-
|-
Line 1,158: Line 1,184:
- PCR7a, 9: false band at 200bp
- PCR7a, 9: false band at 200bp
-
- ccdB: each digestion leads to a right band with ~ 650bp
+
- <font color="#CC33CC">'''ccdB: each digestion leads to a right band with ~ 650bp'''</font>
== 8-28-2010 ==
== 8-28-2010 ==
Line 1,297: Line 1,323:
== 8-31-2010 ==
== 8-31-2010 ==
-
[[Image:31 8 10 apo1.jpg|thumb|right|Gel photo of PCR4a(2.5ng template), PCR4a(5ng template),PCR4b(2.5ng template), PCR4b(5ng template), PCR3(Pfu), PCR3(Phusion)]]
+
[[Image:31 8 10 apo1.jpg|thumb|right|Gel photo of (left to right) PCR4a(2.5ng template), PCR4a(5ng template),PCR4b(2.5ng template), PCR4b(5ng template), PCR3(Pfu), PCR3(Phusion)]]
-
<font color="#009933">Agarose gel electrophoresis of PCR 4a, 4b, 3 (Pfu), 3(Phusion)</font>
+
<font color="#009933">Agarose gel electrophoresis of PCR 4a, PCR4b, PCR3(Pfu), PCR3(Phusion)</font>
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
Line 1,307: Line 1,333:
- results:
- results:
-
- PCR4a(2.5ng template), PCR4a(5ng template),PCR4b(2.5ng template), PCR4b(5ng template), PCR3(Pfu): no band shown
+
- PCR4a(2.5ng template), PCR4a(5ng template),PCR4b(2.5ng template), PCR4b(5ng template), PCR3(Pfu): no band shown
-
- PCR3 (Phusion): right band (~1000bp)
+
- <font color="#CC33CC">'''PCR3 (Phusion): right band (~1000bp)'''</font>
<font color="#009933">New PCR  PCR4a, PCR4b, PCR7a, PCR9</font>
<font color="#009933">New PCR  PCR4a, PCR4b, PCR7a, PCR9</font>
Line 1,315: Line 1,341:
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]]
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]]
 +
PCR mixture for PCR4a, PCR4b
::{|
::{|
|-
|-
-
|PCR4a, PCR4b
+
|template
|37.25 µl (200ng)
|37.25 µl (200ng)
|-
|-
Line 1,334: Line 1,361:
|sum
|sum
|50µl
|50µl
 +
|}
 +
 +
Standard PCR program with annealing temperature PCR4a: 51.1°C, PCR4b: 48.5°C.
 +
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/15 PCR with Phusion| 15 PCR with Phusion]]
 +
 +
PCR mixture for PCR7a, PCR9
 +
::{|
 +
|-
 +
|template
 +
|4 µl (8ng)
 +
|-
 +
|dNTPs
 +
|1  µl
 +
|-
 +
|Phusion
 +
|0.5 µl
 +
|-
 +
|5xbuffer
 +
|10 µl
 +
|-
 +
|DMSO
 +
|1,25µl
 +
|-
 +
|H<sub>2</sub>O
 +
|28.25 µl
 +
|-
 +
|sum
 +
|50µl
 +
|}
 +
 +
PCR program: Phu62
 +
::{|
 +
|-
 +
|98°C
 +
|1 min
 +
|-
 +
|98°C
 +
|10 sec
 +
|-
 +
|62°C
 +
|20 sec
 +
|-
 +
|73°C
 +
|30 sec
 +
|-
 +
|return to step 2 for 29 cycles
 +
|
 +
|-
 +
|73°C
 +
|10 min
 +
|-
 +
|12°C
 +
|forever
 +
|}
 +
 +
 +
[[Image:31 8 10 apo2.jpg|thumb|right|Gel photo of (left to right) PCR3, ladder, PCR7a, empty, PCR9]]
 +
 +
<font color="#009933">Agarose gel electrophoresis of PCR 3, PCR7a, PCR9 for gel extraction</font>
 +
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 +
120V, 30min
 +
 +
- results:
 +
 +
-<font color="#CC33CC"> '''PCR3; right band (~1000bp)'''</font> and side-product
 +
 +
- PCR7a: no band
 +
 +
- PCR9: right band (~800bp) and side-product
 +
 +
<font color="#009933">Gel extraction of the DNA from PCR3 and PCR9</font>
 +
 +
-> Protocol ([[Team:LMU-Munich/Notebook/Protocols/12_Gel_extraction_or_PCR_Clean_up|12 Gel extraction or PCR Clean up]])
 +
 +
results:
 +
 +
PCR 9: 22,5ng/µl; A260/A280=1,8
 +
 +
PCR 3: 22,5ng/µl; A260/A280=2,25
 +
 +
<font color="#009933">New PCR  4a, 4b, 7a with DreamTaq</font>
 +
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/16 PCR with DreamTaq| 16 PCR with DreamTaq]]
 +
 +
PCR mixture for PCR7a
 +
::{|
 +
|-
 +
|template
 +
|5 µl (10ng)
 +
|-
 +
|dNTPs
 +
|5  µl
 +
|-
 +
|DreamTaq
 +
|0.33 µl
 +
|-
 +
|10xbuffer
 +
|5 µl
 +
|-
 +
|DMSO
 +
|1,25µl
 +
|-
 +
|H<sub>2</sub>O
 +
|28.5 µl
 +
|-
 +
|sum
 +
|50µl
 +
|}
 +
 +
Primers for PCR 7a: 13,14
 +
 +
Annealing temp: 60°C
 +
 +
PCR mixture for PCR4a,4b
 +
::{|
 +
|-
 +
|template
 +
|36,5 µl (180ng HeLa cDNA)
 +
|-
 +
|dNTPs
 +
|5  µl
 +
|-
 +
|DreamTaq
 +
|0.33 µl
 +
|-
 +
|10xbuffer
 +
|5 µl
 +
|-
 +
|DMSO
 +
|1,25µl
 +
|-
 +
|sum
 +
|50µl
 +
|}
 +
 +
PCR 4a
 +
 +
Primers for PCR 4a: 7,8
 +
 +
Primers for PCR 4b: 9,10
 +
 +
Annealing temp: 50°C
 +
 +
PCR program:
 +
::{|
 +
|-
 +
|95°C
 +
|1 min
 +
|-
 +
|95°C
 +
|30 sec
 +
|-
 +
|50/60°C
 +
|30 sec
 +
|-
 +
|72°C
 +
|1 min (1kb/min)
 +
|-
 +
|return to step 2 for 29 cycles
 +
|
 +
|-
 +
|72°C
 +
|10 min
 +
|-
 +
|12°C
 +
|forever
|}
|}
== 9-01-2010 ==
== 9-01-2010 ==
-
text
+
 
 +
<font color="#009933">Agarose gel electrophoresis of PCR4a, PCR4b, PCR7 (DreamTaq)</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
150V, 25min
 +
 
 +
results: no product
 +
 
 +
<font color="#009933">New PCR  4a, 4b with DreamTaq, Pfu, with concentration gradient and touch-down PCR </font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/16 PCR with DreamTaq| 16 PCR with DreamTaq]]; [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]]
 +
 
 +
PCR mixture for DreamTaq
 +
::{|
 +
|-
 +
|Concentration
 +
|Low
 +
|Middle
 +
|High
 +
|-
 +
|template
 +
|5 µl (1:1000)
 +
|31.75µl (1:1000)
 +
|2µl (1:10)
 +
|-
 +
|dNTPs
 +
|
 +
|5 µl
 +
|
 +
|-
 +
|DreamTaq
 +
|
 +
|0.33 µl
 +
|
 +
|-
 +
|10xbuffer
 +
|
 +
|5 µl
 +
|
 +
|-
 +
|DMSO
 +
|
 +
|1,25µl
 +
|
 +
|-
 +
|H<sub>2</sub>O
 +
|26.75 µl
 +
|0
 +
|29.75
 +
|-
 +
|sum
 +
|
 +
|50µl
 +
|
 +
|}
 +
 
 +
PCR mixture for Pfu
 +
 
 +
::{|
 +
|-
 +
|Concentration
 +
|Low
 +
|Middle
 +
|High
 +
|-
 +
|template
 +
|5 µl (1:1000)
 +
|37.25µl (1:1000)
 +
|2µl (1:10)
 +
|-
 +
|dNTPs
 +
|
 +
|1 µl
 +
|
 +
|-
 +
|Pfu
 +
|
 +
|0.5 µl
 +
|
 +
|-
 +
|10xbuffer
 +
|
 +
|5 µl
 +
|
 +
|-
 +
|DMSO
 +
|
 +
|1,25µl
 +
|
 +
|-
 +
|H<sub>2</sub>O
 +
|32.25 µl
 +
|0
 +
|35.25 µl
 +
|-
 +
|sum
 +
|
 +
|50µl
 +
|
 +
|}
 +
 
 +
Primers for PCR 4a: 7,8; PCR 4b: 9,10
 +
 
 +
-> Protocol: Thermal cycler program: [[Team:LMU-Munich/Notebook/Protocols/17 Special thermal cycler programs/Touch down|Touch down]]
 +
 
 +
<font color="#009933">Agarose gel electrophoresis of PCR4a, PCR4b</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
150V, 25min
 +
 
 +
[[image: 9.1.10apo.jpg|400px|gel 9-1-10]]
 +
 
 +
from left to right: 4a: P1, P2, P3, D1, D2, D3; 4b: P1, P2, P3, D1, D2, D3
 +
 
 +
key:
 +
 
 +
"P"= PCR with Pfu
 +
 
 +
"D"= PCR with DreamTaq
 +
 
 +
"1"= low template concentration
 +
 
 +
"2"= middle template concentration
 +
 
 +
"3"= high template concentration
 +
 
 +
expected bands:
 +
 
 +
*<font color="#CC33CC">'''4a: 330bp -> P2 and P3 show right bands '''</font>and "primer clouds"(?)
 +
 
 +
*<font color="#CC33CC">'''4b: 376bp -> P1, P2, P3 show right bands'''</font> and "primer clouds" (?)
 +
 
== 9-02-2010 ==
== 9-02-2010 ==
-
text
+
<font color="#009933">Agarose gel electrophorese of PCR 4a P2, 4b P2</font>
-
== 9-05-2010 ==
+
 
-
text
+
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
- 120V, 45min, 1,5% Agarose gel
 +
 
 +
[[Image:2 9 10 apo.jpg|400px|Agarose gel electrophoresis of (from left to right) PCR4aP2, Marker and PCR4bP2]]
 +
 
 +
Agarose gel electrophoresis of (from left to right) PCR4aP2, Marker and PCR4bP2
 +
 
 +
 
 +
- Cut out bands at ~~ 350bp and extract
 +
 
 +
<font color="#009933">PCR Agarose gel extraction</font>
 +
 
 +
-> Protocol:[[Team:LMU-Munich/Notebook/Protocols/14 QIAEX II gel extraction| 14 QIAEX II gel extraction]]
 +
 
 +
results:
 +
 
 +
{|
 +
 
 +
|PCR nr.
 +
|concentration
 +
|A260/A280
 +
|-
 +
 
 +
|4aP2
 +
|12,5 ng/µl
 +
|1,67
 +
|-
 +
 
 +
 
 +
|4bP2
 +
|72,5 ng/µl
 +
|1,53
 +
|-
 +
 
 +
|}
 +
 
 +
 
 +
<font color="#009933">New PCR 7a with Pfu and Phusion</font>
 +
 
 +
template: 190-6, Primer 13,14
 +
 
 +
Mixture with Pfu
 +
 
 +
{|
 +
|-
 +
 
 +
|template (~2ng)
 +
|1µl
 +
|-
 +
 
 +
|Pfu
 +
|0,5µl
 +
|-
 +
 
 +
|Primer *2
 +
|2,5µl *2
 +
|-
 +
 
 +
|10x buffer
 +
|5µl
 +
|-
 +
 
 +
|dNTP Mix
 +
|1µl
 +
|-
 +
 
 +
|DMSO
 +
|1,25µl
 +
|-
 +
 
 +
|H<sub>2</sub>O
 +
|36,25µl
 +
|-
 +
 
 +
|sum
 +
|50µl
 +
|-
 +
|}
 +
 
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]]
 +
 
 +
PCR mixture with Phusion
 +
::{|
 +
|-
 +
|template (~2ng)
 +
|1,0 µl
 +
|-
 +
|dNTPs
 +
|1  µl
 +
|-
 +
|Phusion
 +
|0.5 µl
 +
|-
 +
|5xbuffer
 +
|10 µl
 +
|-
 +
|DMSO
 +
|1,25µl
 +
|-
 +
|H<sub>2</sub>O
 +
|31,25µl
 +
|-
 +
|sum
 +
|50µl
 +
|}
 +
 
 +
-> Protocol:[[Team:LMU-Munich/Notebook/Protocols/15 PCR with Phusion| 15 PCR with Phusion]]
 +
 
 +
<font color="#009933">New PCR 4a, 4b with Pfu</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]]
 +
 
 +
-> Protocol: Thermal cycler program: [[Team:LMU-Munich/Notebook/Protocols/17 Special thermal cycler programs/Touch down|Touch down]]
 +
 
 +
Mixture see 9-1-10, twice 4a and 4b
 +
 
 +
<font color="#009933">Agarose gel electrophorese of PCR 4a, 4b, 7a, gel extracted 4a and 4b</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
- 25min, 150V
 +
 
 +
[[image:9-2-10apo2.jpg|400px|gel 9-2-10-2]]
 +
 
 +
from left to right: 4a*, 4a, 4b*, 4b, 7a Phusion, 7a Pfu, Ladder
 +
 
 +
-> <font color="#CC33CC">'''result: 4b, 4b*: right bands (~330bp)'''</font>
 +
 
 +
-remain: false bands/no band
 +
 
 +
 
 +
[[image:9-2-10apo3.jpg|400px|gel 9-2-10-3]]
 +
 
 +
from left to right: ladder, 4 columns pathway, 4a gelextr., 4b gelextr.
 +
 
 +
-> <font color="#CC33CC">'''results: slight right bands for 4a and 4b'''</font>, no "primer clouds" anymore.
 +
 
 +
== 9-03-2010 ==
 +
 
 +
<font color="#009933">Overlapping PCR 5 with Pfu and Phusion</font>
 +
 
 +
template: 4a, 4b Primer 7,10
 +
 
 +
Mixture with Pfu
 +
 
 +
{|
 +
|-
 +
|PCR4a (~15ng)
 +
|1.2µl
 +
|-
 +
|PCR4b (~15ng)
 +
|2µl (1:10)
 +
|-
 +
|Pfu
 +
|0.5µl
 +
|-
 +
|Primer *2
 +
|2.5µl *2
 +
|-
 +
|10x buffer
 +
|5µl
 +
|-
 +
|dNTP Mix
 +
|1µl
 +
|-
 +
|DMSO
 +
|1,25µl
 +
|-
 +
|H<sub>2</sub>O
 +
|34.05µl
 +
|-
 +
|sum
 +
|50µl
 +
|-
 +
|}
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]]
 +
 
 +
PCR program: standard PCR program for Pfu, Annealing temperature: 54°C
 +
 
 +
PCR mixture with Phusion
 +
::{|
 +
|-
 +
|PCR4a (~15ng)
 +
|1.2µl
 +
|-
 +
|PCR4b (~15ng)
 +
|2µl (1:10)
 +
|-
 +
|dNTPs
 +
|1  µl
 +
|-
 +
|Phusion
 +
|0.5 µl
 +
|-
 +
|5xbuffer
 +
|10 µl
 +
|-
 +
|DMSO
 +
|1,25µl
 +
|-
 +
|H<sub>2</sub>O
 +
|29.05µl
 +
|-
 +
|sum
 +
|50µl
 +
|}
 +
 
 +
-> Protocol:[[Team:LMU-Munich/Notebook/Protocols/15 PCR with Phusion| 15 PCR with Phusion]]
 +
 
 +
PCR program: standard PCR program for Phusion, Annealing temperature: 58°C
 +
 
 +
<font color="#009933">New PCR7a with Pfu</font>
 +
 
 +
template: 190-6; Primer: 13,14
 +
 
 +
Mixture with Pfu
 +
 
 +
{|
 +
|-
 +
|template (~2ng)
 +
|1µl
 +
|-
 +
|Pfu
 +
|0.5µl
 +
|-
 +
|Primer *2
 +
|2.5µl *2
 +
|-
 +
|10x buffer
 +
|5µl
 +
|-
 +
|dNTP Mix
 +
|1µl
 +
|-
 +
|DMSO
 +
|1,25µl
 +
|-
 +
|H<sub>2</sub>O
 +
|36.25µl
 +
|-
 +
|sum
 +
|50µl
 +
|-
 +
|}
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]]
 +
 
 +
PCR program: standard PCR program for Pfu, Gradient: 54.4°C, 57.8°C, 61.4°C, 65.0°C
 +
 
 +
-> results:
 +
 
 +
-7a: only "primer cluods"
 +
 
 +
-5 Pfu: no defined product (slurred?)
 +
 
 +
-5 Phusion: "primer clouds"
 +
 
== 9-04-2010 ==
== 9-04-2010 ==
weekend
weekend
Line 1,348: Line 1,936:
== 9-06-2010 ==
== 9-06-2010 ==
-
text
+
charges for sequencing
 +
{| class="wikitable" border="1"
 +
|-
 +
! name
 +
! 4a-7
 +
! 4a-8
 +
! 4b-9
 +
! 4b-10
 +
! 3-3
 +
! 3-6
 +
! 6-11
 +
! 6-12
 +
|-
 +
| DNA
 +
| 4a; 2.4µl
 +
| 4a; 2.4µl
 +
| 4b; 0.5µl
 +
| 4b; 0.5µl
 +
| 3; 2µl
 +
| 3; 2µl
 +
| 6; 0.5µl
 +
| 6; 0.5µl
 +
|-
 +
| primer [1pmol/µl]
 +
| 7; 3.2µl
 +
| 8; 3.2µl
 +
| 9; 3.2µl
 +
| 10; 3.2µl
 +
| 3; 3.2µl
 +
| 6; 3.2µl
 +
| 11; 3.2µl
 +
| 12; 3.2µl
 +
|-
 +
| Tris (10mM); pH 8.2
 +
| 1.4µl
 +
| 1.4µl
 +
| 3.3µl
 +
| 3.3µl
 +
| 1.8µl
 +
| 1.8µl
 +
| 3.3µl
 +
| 3.3µl
 +
|}
 +
every charge 7µl
 +
 
 +
 
 +
<font color="#009933">New overlapping PCR 5 with Pfu</font>
 +
 
 +
template: 4a, 4b; 15ng
 +
 
 +
Primer 7,10
 +
 
 +
 
 +
 
 +
Mixture
 +
 
 +
{|
 +
|-
 +
|PCR4a (~7ng)
 +
|0.56µl
 +
|-
 +
|PCR4b (~8ng)
 +
|1.1µl (1:10)
 +
|-
 +
|Pfu
 +
|0.5µl
 +
|-
 +
|Primer *2
 +
|2.5µl *2
 +
|-
 +
|10x buffer
 +
|5µl
 +
|-
 +
|dNTP Mix
 +
|1µl
 +
|-
 +
|DMSO
 +
|1,25µl
 +
|-
 +
|H<sub>2</sub>O
 +
|36.6µl
 +
|-
 +
|sum
 +
|50µl
 +
|-
 +
|}
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]]
 +
 
 +
PCR program: standard PCR program for Pfu, Annealing temperature: 46.1°C,48.5°C, 51.1°C
 +
 
 +
-> results: "primer clouds"
 +
 
 +
 
 +
<font color="#009933">New PCR 7a with Pfu</font>
 +
new dilution of 190-6; 1:100
 +
 
 +
{|
 +
|template[2ng/µl]
 +
|1µl
 +
|2µl
 +
|-
 +
|pfu polymerase
 +
|0.5µl
 +
|0.5µl
 +
|-
 +
|primer13
 +
|2.5µl
 +
|2.5µl
 +
|-
 +
|primer14
 +
|2.5µl
 +
|2.5µl
 +
|-
 +
|buffer
 +
|5µl
 +
|5µl
 +
|-
 +
|dNTP Mix
 +
|1µl
 +
|1µl
 +
|-
 +
|DMSO
 +
|1,25µl
 +
|1.25µl
 +
|-
 +
|H<sub>2</sub>O
 +
|36.25µl
 +
|35.25
 +
|-
 +
|sum
 +
|50µl
 +
|50µl
 +
|-
 +
|}
 +
 
 +
 
 +
<font color="#009933">Agarose gel electrophoresis of 7a</font>
 +
 
 +
-> results: "primer clouds"
 +
 
 +
 
 +
<font color="#009933">charges for sequencing</font> for PCR 1,7b and 9
 +
{| class="wikitable" border="1"
 +
|-
 +
! name
 +
! 1-1
 +
! 1-2
 +
! 7b-15
 +
! 7b-16
 +
! 9-20
 +
! 9-21
 +
! 10-22
 +
! 10-23
 +
|-
 +
| DNA
 +
| 1[1:10]; 1.74µl
 +
| 1[1:10]; 1.74µl
 +
| 7b; 3µl
 +
| 7b; 3µl
 +
| 9; 1.78µl
 +
| 9; 1.78µl
 +
| 10; 3.8µl
 +
| 10; 3.8µl
 +
|-
 +
| primer [1pmol/µl]
 +
| 1; 3.2µl
 +
| 2; 3.2µl
 +
| 15; 3.2µl
 +
| 16; 3.2µl
 +
| 20; 3.2µl
 +
| 21; 3.2µl
 +
| 22; 3.2µl
 +
| 23; 3.2µl
 +
|-
 +
| Tris (10mM); pH 8.2
 +
| 2.06µl
 +
| 2.06µl
 +
| 0.8µl
 +
| 0.8µl
 +
| 2.02µl
 +
| 2.02µl
 +
| 0µl
 +
| 0µl
 +
|}
 +
every charge 7µl
 +
 
== 9-07-2010 ==
== 9-07-2010 ==
-
text
+
<font color="#009933">Agarose gel electrophorese of PCR gel extractions</font>
 +
 
 +
150V, 25min
 +
 
 +
-> Protocol:[[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
-result:
 +
 
 +
[[image:7-9-10-1.jpg|400px|gel photo]]
 +
 
 +
from left to right: PCR 1,2a,2b,3,4a,/,7b,9,10,/,ladder,pathway
 +
 
 +
{|
 +
|-
 +
!PCR nr.
 +
!1
 +
!<font color="#CC33CC">2a</font>
 +
!<font color="#CC33CC">2b</font>
 +
!<font color="#CC33CC">3</font>
 +
!<font color="#CC33CC">4a</font>
 +
!<font color="#CC33CC">7b</font>
 +
!9
 +
!<font color="#CC33CC">10</font>
 +
|-
 +
|expected band (bp)
 +
|492
 +
|332
 +
|772
 +
|1087
 +
|330
 +
|402
 +
|808
 +
|1888
 +
|-
 +
|shown band(s)
 +
|550,200
 +
|<font color="#CC33CC">'''300'''</font>
 +
|<font color="#CC33CC">'''750'''</font>
 +
|<font color="#CC33CC">'''1100'''</font>
 +
|<font color="#CC33CC">'''300'''</font>
 +
|<font color="#CC33CC">'''450'''</font>
 +
|900,1500
 +
|<font color="#CC33CC">'''1900'''</font>
 +
|-
 +
|clean charge
 +
|
 +
|<font color="#CC33CC">'''x'''</font>
 +
|<font color="#CC33CC">'''x'''</font>
 +
|<font color="#CC33CC">'''~x'''</font>
 +
|<font color="#CC33CC">'''x'''</font>
 +
|<font color="#CC33CC">'''x'''</font>
 +
|
 +
|<font color="#CC33CC">'''x'''</font>
 +
|-
 +
|}
 +
 
 +
<font color="#009933">new PCR for PCR6</font>
 +
 
 +
PCR mixture for PCR6
 +
 
 +
::{|
 +
|-
 +
|Concentration
 +
|Low
 +
|High
 +
|-
 +
|template
 +
|1 µl (1:100)
 +
|15µl (1:100)
 +
|-
 +
|primer (11,12)
 +
|2.5 µl*2
 +
|
 +
|-
 +
|dNTPs
 +
|1 µl
 +
|
 +
|-
 +
|Pfu
 +
|0.5 µl
 +
|
 +
|-
 +
|10xbuffer
 +
|5 µl
 +
|
 +
|-
 +
|DMSO
 +
|1,25µl
 +
|
 +
|-
 +
|H<sub>2</sub>O
 +
|36.25 µl
 +
|22.25 µl
 +
|-
 +
|sum
 +
|50µl
 +
|
 +
|}
 +
 
 +
-> Protocol: Thermal cycler program: [[Team:LMU-Munich/Notebook/Protocols/17 Special thermal cycler programs/Touch down|Touch down]], 62°C-52°C, 30 cycles by 55°C
 +
 
== 9-08-2010 ==
== 9-08-2010 ==
-
text
+
<font color="#009933">Restriction digestion of eGFP, PCR6, ccdBamp</font>
 +
 
 +
[[Image:Apo_8.9.10.jpg|thumb|right|Gel photo of (left to right) PCR6(5ng/100ng) template]]
 +
 
 +
as prepatation for ligation
 +
 
 +
-> Protocol [[Team:LMU-Munich/Notebook/Protocols/5_Restriction_digest|5 Restriction digestion]]
 +
 
 +
{|
 +
!eGFP
 +
!
 +
!SV40PA=PCR6
 +
!
 +
!ccdBamp
 +
!
 +
|-
 +
|DNA
 +
|5µl=300ng
 +
|DNA
 +
|1µl=37,5ng
 +
|DNA
 +
|2µl=115ng
 +
|-
 +
|Buffer MC
 +
|2µl
 +
|Buffer D
 +
|2µl
 +
 
 +
|Buffer H
 +
|2µl
 +
|-
 +
|BSA 1:10
 +
|2µl
 +
|BSA 1:10
 +
|2µl
 +
|BSA 1:10
 +
|2µl
 +
|-
 +
|EcoRI
 +
|0,5µl
 +
|XbaI
 +
|0,5µl
 +
|EcoRI
 +
|0,5µl
 +
|-
 +
|SpeI
 +
|0,5µl
 +
|PstI
 +
|0,5µl
 +
|PstI
 +
|0,5µl
 +
|-
 +
|H<sub>2</sub>O
 +
|13µl
 +
|H<sub>2</sub>O
 +
|14µl
 +
|H<sub>2</sub>O
 +
|13µl
 +
|-
 +
|sum
 +
|23µl
 +
|sum
 +
|20µl
 +
|sum
 +
|20µl
 +
|-
 +
|}
 +
 
 +
1:30h 37°C, 20min 80°C
 +
 
 +
<font color="#009933">Agarose gel electrophoresis of PCR6</font>
 +
 
 +
-> Protocol:[[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
25min, 150V
 +
 
 +
 
 +
<font color="#009933">Ligation 1</font>
 +
 
 +
{|
 +
|eGFP
 +
|14,4µL
 +
|(144ng)
 +
|-
 +
|SV40PA
 +
|4,8µL
 +
|(48ng)
 +
|-
 +
|ccdBamp
 +
|2,9µL
 +
|(100ng)
 +
|-
 +
|T4 Buffer 10x
 +
|3µL
 +
|
 +
|-
 +
|T4 Ligase
 +
|0,5µL
 +
|
 +
|-
 +
|H<sub>2</sub>O
 +
|4,4µL
 +
|
 +
|-
 +
|sum
 +
|30µL
 +
|
 +
|}
 +
 
 +
 
 +
Ligation at 22,5°C for 30 min, denaturation at 65°C for 10 min.
 +
 
 +
Concentration of DNA in Ligation 1:
 +
 
 +
{|
 +
|540ng
 +
|A260/A280=2,4
 +
|}
 +
 
 +
 
 +
<font color="#009933">Transformation</font>
 +
 
 +
-> Protocol [[Team:LMU-Munich/Notebook/Protocols/18 Competent cells2|18 competent cells2]]
 +
 
 +
The incubation time for the cells is here 1 hour.
 +
 
== 9-09-2010 ==
== 9-09-2010 ==
-
text
+
 
 +
 
 +
<font color="#009933">Agarose gel electrophoresis of PCR 1, 3, 4a, 5, 7a, 9</font>
 +
Agarose gel electrophoresis of PCR 1, 3, 4a, 5, 7a, 9
 +
 
 +
-> protocol [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
results: "primer-clouds", PCR 9: no band
 +
 
 +
 
 +
<font color="#009933">Plasmid extraction of ccdBcam, ccdBamp, Bak, CMV, PhiC31o</font>
 +
 
 +
-> Protocol [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]]
 +
 
 +
{|
 +
|Plasmid
 +
|concentration [ng/µL]
 +
|A260/A280
 +
|-
 +
|ccdBcam
 +
|22.5
 +
|1.0
 +
|-
 +
|ccdBamp
 +
|42.5
 +
|1.2
 +
|-
 +
|Bak
 +
|17.5
 +
|0.8
 +
|-
 +
|CMV
 +
|10.0
 +
|0.7
 +
|-
 +
|PhiC310
 +
|60
 +
|1.4
 +
|}
 +
 
 +
Eluated with H<sub>2</sub>O instead of the Eluation Buffer.
 +
 
 +
 
 +
 
 +
<font color="#009933">New PCR 1, 3, 4a, 5, 7a, 9, 10 with phusion</font>
 +
 
 +
{| class="wikitable" border="1"
 +
|-
 +
! PCRnr.
 +
! 1
 +
! 3
 +
! 4a
 +
! 4b
 +
! 5
 +
! 7a
 +
! 9
 +
! 10
 +
|-
 +
| template
 +
| pDS7 (1:100); 4µl
 +
| 2a; 0.9µl+2b; 0.6µl
 +
| Bak; 0.5µl
 +
| Bak; 0.5µl
 +
| 4a; 0.8µl+4b; 1µl
 +
| 190-6 (1:100); 1µl
 +
| eGFP (1:25); 2µl
 +
| PhiC31o (1:10); 1µl
 +
|-
 +
| primer [10pmol/µl]
 +
| 1,2; 2.5µl
 +
| 3,6; 2.5µl
 +
| 7,8; 2.5µl
 +
| 9,10; 2.5µl
 +
| 7,10; 2.5µl
 +
| 13,14; 2.5µl
 +
| 20,21; 2.5µl
 +
| 22,23; 2.5µl
 +
|-
 +
| H<sub>2</sub>O
 +
| 28µl
 +
| 30.5µl
 +
| 31.5µl
 +
| 31.5µl
 +
| 30.2µl
 +
| 31µl
 +
| 30µl
 +
| 31µl
 +
|-
 +
|Annealing temperature
 +
|51.5°C
 +
|53.1°C
 +
|53.1°C
 +
|51.5°C
 +
|53.1°C
 +
|57.5°C
 +
|61°C
 +
|57.5°C
 +
|}
 +
 
 +
+ in each assay:
 +
 
 +
dNTP mix: 1µl, 5x Phusion buffer: 10µl, DMSO:1.5µl, Phusion:0.5µl
 +
 
 +
sum: 50µl
 +
 
 +
Program: standard Phusion PCR, 29 cycles; annealing temperatures: see above
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/15 PCR with Phusion| 15 PCR with Phusion]]
 +
 
 +
[[Image: apo 09.09 1.jpg|400px|gel electrophoresis of PCR 1, 3, 4a, 5, 7a, 9, 10]]
 +
 
 +
[from left to right: PCR 1, 3, 4a, 5, 7a, 9, 10, Ladder]
 +
 
 +
<font color="#CC33CC">'''PCR3, 9, 10 with right bands.'''</font>
 +
 
 +
<font color="#009933">New PCR 1, 4a, 4b, 5, 7a with phusion</font>
 +
 
 +
{| class="wikitable" border="1"
 +
|-
 +
! PCRnr.
 +
! 1
 +
! 4a
 +
! 4b
 +
! 5
 +
! 7a
 +
|-
 +
| template
 +
| pDS7 (1:10); 1µl
 +
| Bak 1 ; 1µl
 +
| Bak 1; 1µl
 +
| 4a, 4b; 2 x 1.5µl
 +
| pCT 190-6 (1:40); 1µl
 +
|-
 +
| primer
 +
| 1, 2; 2 x 2.5µl
 +
| 7, 8; 2 x 2.5µl
 +
| 9, 10; 2 x 2.5µl
 +
| 7, 10; 2 x 2.5µl
 +
| 13, 14; 2x 2.5µl
 +
|-
 +
| H<sub>2</sub>O
 +
| 31µl
 +
| 31µl
 +
| 31µl
 +
| 30µl
 +
| 30µl
 +
|-
 +
| Annealing temperature
 +
| 52°C
 +
| 52°C
 +
| 48°C
 +
| 50°C
 +
| 55°C
 +
|}
 +
 
 +
+ each assay with dNTP-mix (1µl), 5xBuffer (10µl, DMSO (1.5µl), Phusion (0.5µl)
 +
-> sum: 50µl
 +
 
 +
-> Protocol [[Team:LMU-Munich/Notebook/Protocols/17 Special thermal cycler programs/Touch down phusion|Touch down 59 with phusion]], 30 cycles with gradient appropriate for the annealing temperatures above.
 +
 
== 9-10-2010 ==
== 9-10-2010 ==
-
text
+
<font color="#009933">Agarose gel electrophoresis of PCR 1, 4a, 4b, 5, 7a</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
[[Image: 9-10-10.jpg|400px|gel electrophoresis of PCR 1, 4a, 4b, 5, 7a]]
 +
 
 +
[From left to right: Ladder, 1, 4a, 4b, 5, 7a]
 +
 
 +
Only<font color="#CC33CC"> '''4a has been amplified successfully.'''</font>
 +
 
 +
 
 +
<font color="#009933">Agarose gel extraction of PCR 3, 4a, 5, 9, 10</font>
 +
 
 +
[[Image: apo-10-9-10-2.jpg|400px|gel electrophoresis of PCR 3, 4a, 5, 9, 10]]
 +
 
 +
from left to right:<font color="#CC33CC"> '''PCR 3, band ~700bp, PCR 4a, band ~300bp, PCR 5, bands ~550bp (5*), ~650bp (5), PCR 9, band ~800bp, PCR 10, band ~1900bp'''</font>
 +
 
 +
results:
 +
 
 +
{| class="wikitable" border="1"
 +
|-
 +
!
 +
! PCR 3
 +
! PCR 4a
 +
! PCR 5
 +
! PCR 5*
 +
! PCR 9
 +
! PCR 10
 +
|-
 +
| concentration [ng/µl]
 +
| 20
 +
| 10
 +
| 5
 +
| 30
 +
| 35
 +
| 25
 +
|-
 +
| A260/A280
 +
| 1.6
 +
| 1.333
 +
| 2.0
 +
| 2.0
 +
| 1.750
 +
| 2.0
 +
|}
 +
 
 +
 
 +
 
 +
-> Protocol [[Team:LMU-Munich/Notebook/Protocols/12 Gel extraction or PCR Clean up| 12 Gel extraction or PCR Clean up (Promega kit)]]
 +
 
== 9-11-2010 ==
== 9-11-2010 ==
weekend
weekend
Line 1,363: Line 2,570:
== 9-13-2010 ==
== 9-13-2010 ==
-
text
+
charges for sequencing (retry of 6.9.)
 +
{| class="wikitable" border="1"
 +
|-
 +
! name
 +
! 4a-7
 +
! 4a-8
 +
! 4b-9
 +
! 4b-10
 +
! 3-3
 +
! 3-6
 +
! 6-11
 +
! 6-12
 +
|-
 +
| DNA
 +
| 4a; 2.4µl
 +
| 4a; 2.4µl
 +
| 4b; 0.5µl
 +
| 4b; 0.5µl
 +
| 3; 2µl
 +
| 3; 2µl
 +
| 6; 0.5µl
 +
| 6; 0.5µl
 +
|-
 +
| primer [1pmol/µl]
 +
| 7; 3.2µl
 +
| 8; 3.2µl
 +
| 9; 3.2µl
 +
| 10; 3.2µl
 +
| 3; 3.2µl
 +
| 6; 3.2µl
 +
| 11; 3.2µl
 +
| 12; 3.2µl
 +
|-
 +
| Tris (10mM); pH 8.2
 +
| 1.4µl
 +
| 1.4µl
 +
| 3.3µl
 +
| 3.3µl
 +
| 1.8µl
 +
| 1.8µl
 +
| 3.3µl
 +
| 3.3µl
 +
|}
 +
every charge 7µl
 +
 
 +
 
 +
<font color="#009933">New PCR7a with Taq</font>
 +
 
 +
template: p190-6
 +
 
 +
Primer 13,14
 +
 
 +
Mixture
 +
 
 +
{|
 +
|-
 +
|template (~4ng)
 +
|2µl (1:100)
 +
|-
 +
|MasterMix for Taq
 +
|10µl
 +
|-
 +
|Primer *2
 +
|1.5µl *2
 +
|-
 +
|DMSO
 +
|0,5µl
 +
|-
 +
|H<sub>2</sub>O
 +
|4.5µl
 +
|-
 +
|sum
 +
|20µl
 +
|-
 +
|}
 +
 
 +
PCR program: touchdown PCR with Taq
 +
 
 +
::{|
 +
|1: 94°C 2'
 +
----
 +
|-
 +
|2: 94°C 30"
 +
|-
 +
|3: 64°C/62°C/60°C/58°C/56°C/54° 30"
 +
|-
 +
|4: 72°C 2'
 +
|-
 +
|5: (for each temperature)repeat 2-4 2x
 +
----
 +
|-
 +
|6: 94°C 30"
 +
|-
 +
|7: 58°C 30"
 +
|-
 +
|8: 72°C 2'
 +
|-
 +
|9: repeat 6-8 29x
 +
----
 +
|-
 +
|10: 72°C 10'
 +
|-
 +
|11: 15°C break
 +
|}
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]]
 +
 
 +
<font color="#009933">overnight culture inoculated of</font>
 +
 
 +
:CMV (amp)
 +
:ccdB (amp)
 +
:ccdB (cam)
 +
:pC31o (amp)
 +
:Bak (amp)
 +
 
 +
 
 +
<font color="#009933">New PCR7a with Phusion Hot Start</font>
 +
 
 +
with 2ng and 4 ng template
 +
 
 +
::{|
 +
|-
 +
|template
 +
|2ng
 +
|4ng
 +
|-
 +
|H<sub>2</sub>O
 +
|54µl
 +
|53µl
 +
|-
 +
|Buffer 5x
 +
|20µl
 +
|20µl
 +
|-
 +
|Primer (13,14)
 +
|2*10µl
 +
|2*10µl
 +
|-
 +
|DMSO
 +
|3µl
 +
|3µl
 +
|-
 +
|Hot Start
 +
|1µl
 +
|1µl
 +
|}
 +
 
 +
total: 100µl each, divided into 5 charges
 +
 
 +
program:
 +
 
 +
::98°C 30sec
 +
::----
 +
::98°C 10sec
 +
::gradient: 50°C, 53°C, 56,6°C, 60,2°C, 64,5°C  30sec  30 cycles
 +
::72 15sec
 +
::----
 +
::72°C 5min
 +
::12°C forever
 +
 
 +
-> Protocol:[[Team:LMU-Munich/Notebook/Protocols/20 PCR with Phusion Hot Start|20 PCR with Phusion Hot Start]]
 +
 
== 9-14-2010 ==
== 9-14-2010 ==
-
text
+
<font color="#009933">Agarose gelelectrophoresis of PCR 7a</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
bands 1 to 5: 2ng of template DNA
 +
 
 +
bands 6 to 10: 4ng of template DNA
 +
 
 +
-> no bands
 +
 
 +
<font color="#009933">new PCR 7a and PCR 8 (without mutation)</font>
 +
with mastermix, without DMSO
 +
 
 +
{|
 +
|-
 +
|number:
 +
|7a diluted
 +
|7a undiluted
 +
|8
 +
|-
 +
|mastermix
 +
|50µl
 +
|50µl
 +
|10µl
 +
|-
 +
|primer
 +
|2*10µl
 +
|2*10µl
 +
|2*2µl
 +
|-
 +
|H<sub>2</sub>O
 +
|29µl
 +
|29µl
 +
|5µl
 +
|-
 +
|template
 +
|1µl 190-6 (1:100)
 +
|1µl 190-6
 +
|1µl 190-6 (1:100)
 +
|}
 +
 
 +
7a diluated and undiluated: divided into 3 charges
 +
 
 +
7a diluated: 1,2,3; 7a undiluated: 4,5,6
 +
 
 +
{|
 +
|-
 +
|1
 +
|2
 +
|3
 +
|4
 +
|5
 +
|6
 +
|8
 +
|-
 +
|48°C
 +
|52°C
 +
|56,1°C
 +
|48°C
 +
|52°C
 +
|56,1°C
 +
|52°C
 +
|}
 +
 
 +
program:
 +
 
 +
::{|
 +
|94°C 2'
 +
----
 +
|-
 +
|94°C 30"
 +
|-
 +
|48°C/52°C/56,1°C 30"
 +
|-
 +
|72°C 1,5'
 +
|-
 +
|(for each temperature)repeat 30x
 +
----
 +
|-
 +
|72°C 5'
 +
|}
 +
 
 +
<font color="#009933">Plasmid extraction of ccdB (amp) and ccdB (cam)</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]]
 +
 
 +
results:
 +
 
 +
::ccdB(amp): 105ng/µl A260/280: 2,00
 +
::cddB(cam): 102ng/µl A260/280: 1,952
 +
 
== 9-15-2010 ==
== 9-15-2010 ==
-
text
+
<font color="#009933">Agarose gelelectrophoresis of PCR 7a diluted &7a undiluted & "8"(without mutation)</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
[[Image:9 15 2010.jpg|400px|9 15 10 Gelphoto]]
 +
 
 +
from left to right: 7a: 1:100 diluted: 48°C, 52°C, 56.1°C, undiluted: 48°C, 52°C, 56.1°C, 8 without Mutation
 +
 
 +
<font color="#CC33CC">'''weak right band (with fuzz)for 7a '''</font>undiluated with best result for 56°C
 +
 
 +
-> <font color="#009933">new PCR 7a and "8"</font>
 +
 
 +
with mastermix Taq
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]]
 +
 
 +
template: 190-6, 1:10 and 1:5
 +
 
 +
temp: 56°C and 58°C
 +
 
 +
 
 +
{|
 +
|-
 +
|number
 +
|7a
 +
|7a
 +
|"8"
 +
|"8"
 +
|-
 +
|Mastermix
 +
|20µl
 +
|20µl
 +
|20µl
 +
|20µl
 +
|-
 +
|H<sub>2</sub>O
 +
|15µl
 +
|15µl
 +
|15µl
 +
|15µl
 +
|-
 +
|Primer
 +
|(13,14) 2*2µl
 +
|(13,14) 2*2µl
 +
|(13,16) 2*2µl
 +
|(13,16) 2*2µl
 +
|-
 +
|Template
 +
|190-6 (1:5) 1µl
 +
|190-6 (1:10) 1µl
 +
|190-6 (1:5) 1µl
 +
|190-6 (1:10) 1µl
 +
|}
 +
 
 +
2 charges each: one for 56°C and one for 58°C annealing temperature
 +
 
 +
 
 +
program:
 +
 
 +
::{|
 +
|94°C 2'
 +
----
 +
|-
 +
|94°C 30"
 +
|-
 +
|56°C/58°C 30"
 +
|-
 +
|72°C 1,5'
 +
|-
 +
|(for each temperature)repeat 30x
 +
----
 +
|-
 +
|72°C 5'
 +
|}
 +
 
 +
 
 +
<font color="#009933">Restriction Digestion of PCR 1 (17.8.), PCR 3 (10.9.), PCR 5<sub>1</sub> (10.9.), PCR 5<sub>2</sub> (10.9.), PCR 6 (17.8.), PCR 9 (10.9.), PCR 10 (30.8.) for ligation with vector</font>
 +
 
 +
EcoR1 + Pst1 with Buffer H; 50ng DNA
 +
 
 +
{|
 +
|
 +
|1
 +
|3
 +
|5<sub>1</sub>
 +
|5<sub>2</sub>
 +
|6
 +
|9
 +
|10
 +
|-
 +
|H<sub>2</sub>O
 +
|13µl
 +
|12,5µl
 +
|5µl
 +
|13µl
 +
|13,5µl
 +
|13,5µl
 +
|12,5µl
 +
|-
 +
|Buffer H
 +
|2µl
 +
|2µl
 +
|2µl
 +
|2µl
 +
|2µl
 +
|2µl
 +
|2µl
 +
|-
 +
|BSA (1:10)
 +
|2µl
 +
|2µl
 +
|2µl
 +
|2µl
 +
|2µl
 +
|2µl
 +
|2µl
 +
|-
 +
|DNA
 +
|2µl
 +
|2,5µl
 +
|10µl
 +
|2µl
 +
|1,5µl
 +
|1,5µl
 +
|2,5µl
 +
|-
 +
|EcoR1
 +
|0,5µl
 +
|0,5µl
 +
|0,5µl
 +
|0,5µl
 +
|0,5µl
 +
|0,5µl
 +
|0,5µl
 +
|-
 +
|Pst1
 +
|0,5µl
 +
|0,5µl
 +
|0,5µl
 +
|0,5µl
 +
|0,5µl
 +
|0,5µl
 +
|0,5µl
 +
|}
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/5 Restriction digest|5 Restriction digestion]]
 +
 
 +
<font color="#009933">Agarose gelelectrophoresis of PCR 7a 1-4 & "8" 1-4(without mutation)</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
-> bad results
 +
 
 +
<font color="#009933">purification of restriction digestion</font>
 +
 
 +
{|
 +
|-
 +
|
 +
|concentration (ng/µl)
 +
|A<sub>260</sub>/A<sub>280</sub>
 +
|(supposed) lenght
 +
|-
 +
|1
 +
|20
 +
|1,000
 +
|492
 +
|-
 +
|3
 +
|5
 +
|1,000
 +
|1087
 +
|-
 +
|5<sub>1</sub>
 +
|22,5
 +
|1,8
 +
|688
 +
|-
 +
|5<sub>2</sub>
 +
|7,5
 +
|1,5
 +
|688
 +
|-
 +
|6
 +
|10
 +
|1,333
 +
|237
 +
|-
 +
|9
 +
|20
 +
|1,6
 +
|808
 +
|-
 +
|10
 +
|17,5
 +
|1,75
 +
|1888
 +
|}
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/12 Gel extraction or PCR Clean up| 12 Gel extraction or PCR Clean up (Promega kit)]]
 +
 
 +
<font color="#009933">Ligation with pSB1C3 (2072bp)</font>
 +
 
 +
{|
 +
|-
 +
|for
 +
|#
 +
|backbone
 +
|insert (µl)
 +
|charge (µl)
 +
|Buffer 10x (µl)
 +
|H<sub>2</sub>O (µl)
 +
|-
 +
|100ng
 +
|1
 +
|4
 +
|7,12
 +
|20
 +
|2
 +
|6,38
 +
|-
 +
|50ng
 +
|3
 +
|2
 +
|31,48
 +
|40
 +
|4
 +
|2,02
 +
|-
 +
|100ng
 +
|5<sub>1</sub>
 +
|4
 +
|8,85
 +
|20
 +
|2
 +
|4,65
 +
|-
 +
|100ng
 +
|5<sub>2</sub>
 +
|4
 +
|26,56
 +
|40
 +
|4
 +
|4,94
 +
|-
 +
|100ng
 +
|6
 +
|4
 +
|6,86
 +
|20
 +
|2
 +
|13,36
 +
|-
 +
|100ng
 +
|9
 +
|4
 +
|11,7
 +
|20
 +
|2
 +
|1,8
 +
|-
 +
|100ng
 +
|10
 +
|4
 +
|31,24
 +
|40
 +
|4
 +
|4,26
 +
|}
 +
plus 1 µl T4 ligase in each charge
 +
 
 +
-> Protocol:  [[Team:LMU-Munich/Notebook/Protocols/22 Ligation|22 Ligation]]
 +
 
 +
 
 +
<font color="#009933">new PCR 7a & "8"</font>
 +
 
 +
with mastermix Taq 56°C
 +
 
 +
{|
 +
|-
 +
|
 +
|7a-1
 +
|7a-2
 +
|"8"-1
 +
|"8"-2
 +
|-
 +
|mastermix (µl)
 +
|10
 +
|10
 +
|10
 +
|10
 +
|-
 +
|template
 +
|190-6 1µl
 +
|190-6 2µl
 +
|190-6(1:10) 1µl
 +
|190-6(1:10) 2µl
 +
|-
 +
|primer
 +
|13&14 2*1µl
 +
|13&14 2*1µl
 +
|13&16 2*1µl
 +
|13&16 2*1µl
 +
|-
 +
|H<sub>2</sub>O
 +
|7µl
 +
|6µl
 +
|7µl
 +
|6µl
 +
|}
 +
sum: 20µl each
 +
 
 +
pcr-program:
 +
::{|
 +
|94°C 2'
 +
----
 +
|-
 +
|94°C 30"
 +
|-
 +
|56°C 30"
 +
|-
 +
|72°C 1,5'
 +
|-
 +
|(for each temperature)repeat 30x
 +
----
 +
|-
 +
|72°C 5'
 +
|-
 +
|12°C forever
 +
|}
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]]
 +
 
== 9-16-2010 ==
== 9-16-2010 ==
-
text
+
<font color="#009933">Agarose gelelectrophoresis of PCR 7a-1, PCR 7a-2, PCR 8-1, 8-2 and of all three PCR 6 we ever made (in order to control whether we have extracted the right fragment (237) or just the primerdimers)</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
[[Image:9 16 10.jpg|400px|9 16 10 Gelphoto]]
 +
 
 +
From left to right: 6<sub>2</sub>, 6<sub>1</sub>, 6, Ladder, 7a-1, 7a-2, 8-2, 8-1
 +
 
 +
::results:
 +
 
 +
::6-1 and 6-2: ?
 +
 
 +
::<font color="#CC33CC">'''6: ok'''</font>
 +
::7a-1,7a-2,"8"-1,"8"-2: primerdimer-problem -> we ordered new primers!
 +
 
 +
<font color="#009933">transformation of the ligations 1,3,5<sub>1</sub>, 5<sub>2</sub>, 6, 9, 10</font>
 +
 
 +
-> Protocol:[[Team:LMU-Munich/Notebook/Protocols/3 Transformation|3 Transformation]]
 +
 
 +
->plated on plates with chloramphenicol
 +
 
 +
<font color="#009933">again PCR 1,3,5,6,9,10 in order to increase the profit (Ausbeute) for ligation</font>
 +
 
 +
{|
 +
|-
 +
|
 +
|1
 +
|3
 +
|5
 +
|6
 +
|9
 +
|10
 +
|-
 +
|template
 +
|prev TRE(1:30) 1µl
 +
|2a(1:5) 1µl + 2b 0,5µl
 +
|4a 0,5µl + 4b 0,5µl
 +
|SV40PA (pcDNA3)(1:50) 1µl
 +
|eGFP(1:5) 1µl
 +
|PhiC310(1:6) 1µl
 +
|-
 +
|primer
 +
|1&2 2*2,5µl
 +
|3&6 2*2,5µl
 +
|7&10 2*2,5µl
 +
|11&12 2*2,5µl
 +
|20&21 2*2,5µl
 +
|22&23 2*2,5µl
 +
|-
 +
|T<sub>m</sub>
 +
|54°C
 +
|50°C
 +
|49°C
 +
|56°C
 +
|58°C
 +
|58°C
 +
|-
 +
|mastermix (µl)
 +
|25
 +
|25
 +
|25
 +
|25
 +
|25
 +
|25
 +
|-
 +
|DMSO
 +
|1,25µl
 +
|1,25µl
 +
|1,25µl
 +
|1,25µl
 +
|1,25µl
 +
|1,25µl
 +
|-
 +
|H<sub>2</sub>O (µl)
 +
|17,75
 +
|17,25
 +
|17,75
 +
|17,75
 +
|17,75
 +
|17,75
 +
|}
 +
 
 +
pcr-program:
 +
 
 +
::{|
 +
|94°C 2'
 +
----
 +
|-
 +
|94°C 30"
 +
|-
 +
|T<sub>m</sub> (see above) 30"
 +
|-
 +
|72°C 1,5'
 +
|-
 +
|(for each temperature)repeat 30x
 +
----
 +
|-
 +
|72°C 5'
 +
|}
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]]
 +
 
== 9-17-2010 ==
== 9-17-2010 ==
-
text
+
<font color="#009933">Analysis of the transformation from yesterday</font>
 +
 
 +
Colonies on plates when 100µl or pellet plated:
 +
 
 +
{|
 +
|-
 +
|
 +
|1
 +
|3
 +
|5
 +
|6
 +
|9
 +
|10
 +
|-
 +
|pellet
 +
|no
 +
|yes
 +
|yes
 +
|yes
 +
|yes
 +
|yes
 +
|yes
 +
|-
 +
|100µl
 +
|no
 +
|yes
 +
|yes
 +
|yes
 +
|yes
 +
|yes
 +
|yes
 +
|}
 +
 
 +
<font color="#009933">again: transformation of ligation 1</font>
 +
 
 +
->Protocol: [[Team:LMU-Munich/Notebook/Protocols/3 Transformation|3 Transformation]]
 +
 
 +
<font color="#009933">Agarose gelelectrophoresis of yesterday's PCR 1,3,5,6,9,10(without mutation)</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
[[Image:9 17 10apo.jpg| 400px |9 17 10 Gelphoto]]
 +
 
 +
From left to right: PCR1, PCR3, PCR5, Ladder, PCR6, PCR9, PCR10
 +
 
 +
-> <font color="#CC33CC">'''right band for PCR 3,5,6,9,10'''</font>; no band for PCR 1
 +
 
 +
[[Image:9 17 10apo2.jpg| 400px |9 17 10 Gelphoto2]]
 +
 
 +
From left to right: Ladder, PCR3, PCR5, PCR9, PCR10
 +
 
 +
Bands took: 3<sub>2</sub>: Band short over 1000bp, 3<sub>1</sub>: Band short under 1000bp (probably 3<sub>2</sub> right), 5 the highest band, 9 upper band, 10 upper band
 +
 
 +
<font color="#009933">new PCR 1,5,6</font>
 +
 
 +
{|
 +
|-
 +
|
 +
|1
 +
|5
 +
|6
 +
|-
 +
|template
 +
|pTRE Rev 0,5µl
 +
|4a 1,5µl + 4b 1,5µl
 +
|pcDNA3 0,5µl
 +
|-
 +
|primer
 +
|1&2 2*2,5µl
 +
|7&10 2*2,5µl
 +
|11&12 2*2,5µl
 +
|-
 +
|buffer 10x
 +
|5µl
 +
|5µl
 +
|5µl
 +
|-
 +
|dNTPs
 +
|1µl
 +
|1µl
 +
|1µl
 +
|-
 +
|Pfu
 +
|0,5µl
 +
|0,5µl
 +
|0,5µl
 +
|-
 +
|H<sub>2</sub>O
 +
|26,75 µl
 +
|24,25 µl
 +
|26,75 µl
 +
|-
 +
|T<sub<m</sub>
 +
|51°C
 +
|50°C
 +
|55°C
 +
|}
 +
 
 +
-> Protocol:
 +
pcr-program: standard Pfu [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]]
 +
 
 +
 
 +
<font color="#009933">Restriction digestion of PCR products, ccdB Plasmids and Biobricks for the 3A Method</font>
 +
 
 +
 
 +
{|
 +
|Name
 +
|H<sub>2</sub>O
 +
|Buffer (each 2µl)
 +
|BSA (1:10)
 +
|DNA Volume
 +
|DNA Mass
 +
|Enzyms (2*0.5µl)
 +
|-
 +
|ccdBa
 +
|25 µl
 +
|H
 +
|2µl
 +
|20µl
 +
|~600ng
 +
|E+P
 +
|-
 +
|ccdBa
 +
|25µl
 +
|H
 +
|2µl
 +
|20µl
 +
|~700ng
 +
|E+P
 +
|-
 +
|1
 +
|43µl
 +
|MC
 +
|2µl
 +
|2µl
 +
|~500ng
 +
|E+S
 +
|-
 +
|3
 +
|25µl
 +
|B
 +
|2µl
 +
|20µl
 +
|~200ng
 +
|X+P
 +
|-
 +
|Primer 18+19
 +
|25µl
 +
|B
 +
|2µl
 +
|10µl+10µl
 +
|?
 +
|X+P
 +
|-
 +
|5<sub>1</sub>
 +
|25µl
 +
|MC
 +
|2µl
 +
|20µl
 +
|~100ng
 +
|E+S
 +
|-
 +
|5<sub>2</sub>
 +
|25µl
 +
|MC
 +
|2µl
 +
|20µl
 +
|~600ng
 +
|E+S
 +
|-
 +
|6
 +
|35µl
 +
|B
 +
|2µl
 +
|10µl
 +
|~400ng
 +
|X+P
 +
|-
 +
|9
 +
|25µl
 +
|MC
 +
|2µl
 +
|20µl
 +
|~700ng
 +
|E+S
 +
|-
 +
|10
 +
|25µl
 +
|MC
 +
|2µl
 +
|20µl
 +
|~400ng
 +
|E+S
 +
|-
 +
|eGFP
 +
|35µl
 +
|MC
 +
|2µl
 +
|10µl
 +
|~550ng
 +
|E+S
 +
|-
 +
|CMV1
 +
|35µl
 +
|H
 +
|2µl
 +
|10µl
 +
|~530ng
 +
|E+P
 +
|}
 +
 
 +
-> Protocol:[[Team:LMU-Munich/Notebook/Protocols/5 Restriction digest|5 Restriction digestion]]
 +
 
 +
<font color="#009933">Colony PCR of Ligations 3, 5<sub>1</sub>, 5<sub>2</sub>, 6, 9, 10 and PCR of CMV1 (to test if right)</font>
 +
 
 +
10 1-4, 9 1-4, 6 1-4, 5<sub>1</sub> 1-4, 5<sub>2</sub> 1-4, 3 1-4 Colonies picked and put in following Mix:
 +
 
 +
{|
 +
|PCR Mastermix
 +
|10µl
 +
|-
 +
|Biobrick Primer F
 +
|1.5µl
 +
|-
 +
|Biobrick Primer R
 +
|1.5µl
 +
|-
 +
|H<sub>2</sub>O
 +
|7µl
 +
|}
 +
 
 +
CMV1:
 +
 
 +
{|
 +
|PCR Mastermix
 +
|10µl
 +
|-
 +
|Biobrick Primer F
 +
|1.5µl
 +
|-
 +
|Biobrick Primer R
 +
|1.5µl
 +
|-
 +
|Template
 +
|2µl
 +
|-
 +
|H<sub>2</sub>O
 +
|5µl
 +
|}
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]]
 +
 
 +
<font color="#009933">Agarose Gel electrophoresis of 3, 5<sub>1</sub>, 5<sub>2</sub>, 6, 9, 10 and CMV1 (to test if right)</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
[[Image:17 9 10apo.jpg|400px|17 9 10 Gelphoto]]
 +
 
 +
Above: From left to right: 3.1, 3.2, 3.3, 3.4, 5<sub>1</sub>.1, 5<sub>1</sub>.2, 5<sub>1</sub>.3, 5<sub>1</sub>.4, Ladder, 5<sub>2</sub>.1, 5<sub>2</sub>.2, 5<sub>2</sub>.3, 5<sub>2</sub>.4
 +
 
 +
Below: From left to right: 6.1, 6.2, 6.3, 6.4, 9.1, 9.2, 9.3, 9.4, Ladder, 10.1, 10.2, 10.3, 10.4, CMV1
 +
 
 +
-> <font color="#CC33CC">'''right bands for 6.3 and 6.4, CMV1 ~1200bp (we think that this is right, as Biobrick sequenzing information indicates that it isn't 654bp but about 1200bp)'''</font>
 +
 
 +
 
 +
<font color="#009933">PCR clean up of PCR Gel extraction 3<sub>1</sub>, 3<sub>2</sub>, 5, 9, 10, PCR Product 6 and digestion PCR1, Primer 18+19, R5<sub>1</sub>, R5<sub>2</sub>, PCR6, PCR9, PCR10, PCR3</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/12 Gel extraction or PCR Clean up| 12 Gel extraction or PCR Clean up (Promega kit)]]
 +
 
== 9-18-2010 ==
== 9-18-2010 ==
weekend
weekend
Line 1,378: Line 3,536:
== 9-20-2010 ==
== 9-20-2010 ==
-
text
+
<font color="#009933">Dephosphorylation of linearized ccdB amp and ccdB cam</font>
 +
 
 +
Add 1µl TSAP to digested vectors.
 +
 
 +
Incutation: 37°C 15min; Inhibition: 74°C 15min
 +
 
 +
<font color="#009933">Ligation and 3A-Assemblies</font>
 +
 
 +
Jump-or-Die Ligations: 1a, 1b (5<sub>1</sub>),1b  (5<sub>2</sub>), 2, 3
 +
 
 +
Cut'N'Survive Ligations: 1a, 2b,
 +
 
 +
Biobricks for both Systems: CMV, PCR1 (tet-on-promoter)
 +
 
 +
Mixtures:
 +
 
 +
- 3A Assemblies (everything except CMV, PCR1):
 +
 
 +
Inserts: each 8µl; ccdB vector: 1µl; T4 Ligase: 0.5µl; T4 Ligase Buffer: 2µl; H<sub>2</sub>O: 1µl
 +
 
 +
- Biobricks:
 +
 
 +
Insert: 10µl; ccdB vector: 1µl; T4 Ligase: 0.5µl; T4 Ligase Buffer: 2µl; H<sub>2</sub>O: 7µl
 +
 
 +
Incubation: 2:30h 16°C; Inhibition: 10 min 65°C
 +
 
 +
->Protocols: [[Team:LMU-Munich/Notebook/Protocols/22 Ligation|22 Ligation]], [[Team:LMU-Munich/Notebook/Protocols/13 3A Method for Biobrick assembly| 13 3A Method for Biobrick assembly]]
 +
 
 +
<font color="#009933">Colony PCR of PCR product ligations with pSB1C3</font>
 +
 
 +
Mixture:
 +
 
 +
PCR Mastermix:130µl ; PrimerF:19,5µl ; PrimerR:19,5µl ; H<sub>2</sub>O:91µl
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]]
 +
 
 +
<font color="#009933">Agarose Gel Electrophoresis of Colony PCRs</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
[[Image:20.9.10.jpg|400px|Colony PCRs]]
 +
 
 +
above from left to right: pathway/ladder/3.5/3.6/3.7/3.8/5<sub>1</sub>.5/5<sub>1</sub>.6/5<sub>1</sub>.7/5<sub>1</sub>.8/5<sub>2</sub>.5/5<sub>2</sub>.6/5<sub>2</sub>.7/5<sub>2</sub>.8
 +
 
 +
below from left to right: ladder/6.5/6.6/6.7/6.8/9.5/9.6/9.7/9.8/10.5/10.6/10.7/10.8
 +
 
== 9-21-2010 ==
== 9-21-2010 ==
-
text
+
<font color="#009933">Agarose Gel Electrophoresis of PCRs 1, 5, 6, and Colony PCR 3.8, 5<sub>1</sub>.7,  5<sub>2</sub>.6, 9.7, 10.5, 10.6</font>
 +
 
 +
-> Bad results, probably too much cells in PCR mix
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
<font color="#009933">Colony PCR of 3.8, 5<sub>1</sub>.7,  5<sub>2</sub>.6, 9.7, 10.5, 10.6 again and of the 3A Ligations of yesterday as well as the Ligation of PCR1 and CMV with pSB1C3</font>
 +
 
 +
PCR Master Mix: 60µl
 +
 
 +
Primer F: 9µl
 +
 
 +
Primer R: 9µl
 +
 
 +
H<sub>2</sub>O: 42µl
 +
 
 +
->22charges à 5µl
 +
 
 +
-> Protocol:[[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]]
 +
 
 +
 
 +
<font color="#009933">PCR 7a with new Primers</font>
 +
 
 +
{|
 +
|-
 +
|template 190-6 (1:10)
 +
|1µl
 +
|-
 +
|Master Mix
 +
|25µl
 +
|-
 +
|Primer 13k (short)
 +
|3,75µl
 +
|-
 +
|Primer 14
 +
|3,75µl
 +
|-
 +
|H<sub>2</sub>O
 +
|16,5µl
 +
|}
 +
 
 +
sum: 50µl
 +
 
 +
same with Primer 13l (long)
 +
 
 +
->Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]]
 +
 
 +
<font color="#009933">Agarose Gel Electrophoresis of Colony PCRs and PCR 7a</font>
 +
different ladder: [[image:fermentas ladder mix.jpg|thumb|right|fermentas dna ladder mix (source:http://www.fermentas.com/en/products/all/dna-electrophoresis/generuler-dna-ladders/sm0333-generuler-mix]]
 +
[[image:21 9 10apo2.jpg|400px|gelfoto 21 9 10 apo2]]
 +
 
 +
 
 +
key: JD=Jump-or-Die; CS=Cut'N'Survive; first number=Ligation number (see schedule); second number=colonie number (marked on the plate)
 +
 
 +
from left to right:7ak(short primer), 7al(long primer), JD.1a.1, JD.1a.2,JD.1b5<sub>1</sub>.1, JD.1b5<sub>2</sub>.2, JD.1b5<sub>2</sub>.1, JD.1b5<sub>2</sub>.2, JD.2.1, JD.2.2, JD.3<sub>1</sub>.1, JD.3<sub>1</sub>.2, CS.1a.1, CS.1a.2
 +
 
 +
 
 +
<font color="#CC33CC">'''verified products:7a (~800bp); JD.2.1; JD.2.2(each ~1000bp);JD.3<sub>1</sub>.1(~2000bp); CS.1a.2(~1500bp)'''</font>
 +
 
 +
no bands shown: CS.2b.1, CS.2b.2, PCR1.1, PCR1.CMV,3.8, 5<sub>1</sub>.7, 5<sub>2</sub>.6, 9.7, 10.5, 10.6
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
== 9-22-2010 ==
== 9-22-2010 ==
-
text
+
<font color="#009933">Inoculate Colonies</font>
 +
 
 +
inoculated in 4ml LB-medium with appropriate antibiotic
 +
 
 +
<font color="#009933">Plated residual transformated E. colis (9-20-10) (where we had few colonies on plates) </font>
 +
 
 +
<font color="#009933">Colony PCRs</font>
 +
 
 +
for 22*5µl charges:
 +
 
 +
{|
 +
|PCR Mastermix
 +
|55µl
 +
|-
 +
|Primer F
 +
|8,25µl
 +
|-
 +
|Primer R
 +
|8,25µl
 +
|-
 +
|H<sub>2</sub>O
 +
|38,5µl
 +
|-
 +
|sum
 +
|110µl
 +
|}
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]]
 +
 
 +
Annealing temperature: 53°C
 +
 
 +
 
 +
<font color="#009933">PCR Purification of 7a</font>
 +
 
 +
charges 7ak and 7al had not been labeled, so they were renamed.
 +
 
 +
results:
 +
 
 +
7a<sub>1</sub>: 135ng/µl; A260/A280=1.7
 +
 
 +
7a<sub>2</sub>: 133ng/µl; A260/A280=1.7
 +
 
 +
 
 +
<font color="#009933">PCR8 with Phusion Hot Start II</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/20 PCR with Phusion Hot Start|20 PCR with Phusion Hot Start]]
 +
 
 +
{|
 +
|-
 +
|template 7a 1:100
 +
|3µl ~4ng (850bp)
 +
|-
 +
|template 7b 1:10
 +
|2µl ~2ng (402bp)
 +
|-
 +
|Buffer 5X
 +
|10µl
 +
|-
 +
|Phusion
 +
|0.5µl
 +
|-
 +
|H<sub>2</sub>O
 +
|27µl
 +
|-
 +
|Primer 13
 +
|2.5µl
 +
|-
 +
|Primer 16
 +
|2.55µl
 +
|-
 +
|DMSO)
 +
|1.5µl
 +
|-
 +
|dNTP mix
 +
|1µl
 +
|}
 +
 
 +
Program: annealing temperature: 63°C, elongation time: 40sec
 +
 
 +
<font color="#009933">Agarose gel electrophoresis of Colony PCRs and PCR8</font>
 +
 
 +
new ladder: fermentas FastRuler (TM) Middle Range DNA Ladder http://www.fermentas.com/en/products/all/dna-electrophoresis/fastruler-dna-ladders/sm111-fastruler-middle
 +
 
 +
[[image: 9 22 10 beschriftet.jpg|400px|gelfoto]]
 +
 
 +
some results:
 +
 
 +
PCR 8 (lanes 23, 24): no band
 +
 
 +
Colony PCRs CS.2b.10, PCR1cam.3, PCR1cam.4, CMVcam.4, CMVcam.4, JD.1b5<sub>1</sub>.10  (lanes 7,8,9,10,11,14): show right bands
 +
 
 +
 
 +
 
 +
 
 +
<font color="#009933">Plasmid extraction of "over-day" cultures ligation-7(=BB4),-8(=BB4),-9(=BB5),-12(=BB9) and pSB1C3</font>
 +
 
 +
->Protocol: [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]]
 +
 
 +
concentrations (ng/µl - A<sub>260/280</sub>):
 +
::BB4(7):57,5 - 1,643
 +
::BB4(8):15 - 1,5
 +
::BB5(9):173 - 1,816
 +
::BB7(12):55,0 - 1,571
 +
::pSB1C3: 12,5 - 1,250
 +
 
 +
<font color="#009933">new PCR 7b in order to get better results</font>
 +
{|
 +
|-
 +
|Master Mix
 +
|25µl
 +
|-
 +
|H<sub>2</sub>O
 +
|16,5µl
 +
|-
 +
|Primer 15
 +
|3,75µl
 +
|-
 +
|Primer 16
 +
|3,75µl
 +
|-
 +
|template 190-6 (1:10)
 +
|1µl
 +
|}
 +
 
 +
<font color="#009933">overnight-cultures</font>
 +
 
 +
inoculated of: CS2bK10=BB11; PCP1K3+4=BB1; CMV-C-K3+4; JD1b5<sub>1</sub>K10=BB2; pSB1C3
 +
 
== 9-23-2010 ==
== 9-23-2010 ==
-
text
+
<font color="#009933">Restriction digestion of BB5 for the 3A Method</font>
 +
 
 +
{|
 +
|Name
 +
|H<sub>2</sub>O
 +
|Buffer D
 +
|BSA (1:10)
 +
|DNA Volume
 +
|DNA Mass
 +
|Enzyms (2*0.5µl)
 +
|-
 +
|BB5
 +
|11 µl
 +
|2µl
 +
|2µl
 +
|4µl
 +
|~500ng
 +
|X+P
 +
|-
 +
|}
 +
 
 +
sum: 20µl
 +
 
 +
-> Protocol:[[Team:LMU-Munich/Notebook/Protocols/5 Restriction digest|5 Restriction digestion]]
 +
 
 +
<font color="#009933">Plasmid extraction of overnight cultures BB2, BB11, CMV in cam and pSB1C3</font>
 +
 
 +
->Protocol: [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]]
 +
 
 +
concentrations (ng/µl - A<sub>260/280</sub>):
 +
::BB2:232 - 1,603
 +
::BB11:170 - 1,889
 +
::CMV in cam:272 - 1,703
 +
::pSB1C3: 290 - 1,758
 +
 
 +
The pSB1C3 colony was pink (produced a red pigment or something like that, so we make a new transformation in order to extract only pSB1C3 and not some other vectors.
 +
 
 +
<font color="#009933">Agarose gelelectrophoresis of PCR 7b and pathway restriction digestions</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
[[Image:23 9 10 1.jpg|thumb|right|Gelphoto]]
 +
 
 +
From left to right: PCR7b, pathway (3x),ladder (5000,2000,850,400,100bp)
 +
 
 +
result: <font color="#CC33CC">'''PCR 7b shows right band with ~400bp '''</font>
 +
 
 +
<font color="#009933">PCR clean up of PCR 7b</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/12 Gel extraction or PCR Clean up| 12 Gel extraction or PCR Clean up (Promega kit)]]
 +
 
 +
result: 90ng/µl; A260/A280= 1,895
 +
 
 +
 
 +
 
 +
<font color="#009933">Restriction digestion of BB2 and BB11</font>
 +
 
 +
 
 +
{|
 +
|Name
 +
|H<sub>2</sub>O
 +
|Buffer D
 +
|BSA (1:10)
 +
|DNA Volume
 +
|DNA Mass
 +
|Enzyms (2*0.5µl)
 +
|-
 +
|BB2
 +
|10 µl
 +
|2µl
 +
|2µl
 +
|5µl
 +
|~1.2mg
 +
|X+P
 +
|-
 +
|}
 +
 
 +
sum: 20µl
 +
 
 +
 
 +
{|
 +
|Name
 +
|H<sub>2</sub>O
 +
|Buffer D
 +
|BSA (1:10)
 +
|DNA Volume
 +
|DNA Mass
 +
|Enzyms (2*0.5µl)
 +
|-
 +
|BB11
 +
|7 µl
 +
|2µl
 +
|2µl
 +
|8µl
 +
|~1.3mg
 +
|X+P
 +
|-
 +
|}
 +
 
 +
sum: 20µl
 +
 
 +
-> Protocol [[Team:LMU-Munich/Notebook/Protocols/5 Restriction digest|5 Restriction digestion]]
 +
 
 +
 
 +
<font color="#009933">Ligation of BB1 and BB6</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/22 Ligation|22 Ligation]]
 +
 
 +
{|
 +
|Name
 +
|H<sub>2</sub>O
 +
|T4 Ligase-Buffer
 +
|PCR1
 +
|Primer 18+19
 +
|ccdBAmp
 +
|T4 Ligase
 +
|-
 +
|BB1
 +
|9.5 µl
 +
|2µl
 +
|5µl
 +
|2µl
 +
|1µl
 +
|0.5µl
 +
|-
 +
|}
 +
 
 +
{|
 +
|Name
 +
|H<sub>2</sub>O
 +
|T4 Ligase-Buffer
 +
|CMV E+S
 +
|BB5
 +
|ccdBCam
 +
|T4 Ligase
 +
|-
 +
|BB6
 +
|6.5 µl
 +
|2µl
 +
|8µl
 +
|2µl
 +
|1µl
 +
|0.5µl
 +
|-
 +
|}
 +
 
 +
<font color="#009933">PCR 8 with 7b(new) plus 7a-2 with Phusion Hot Start II; Primer 16 plus 13k and 13l</font>
 +
 
 +
{|
 +
|-
 +
|Buffer 5x
 +
|10µl
 +
|-
 +
|H<sub>2</sub>O
 +
|27µl
 +
|-
 +
|dNTPs
 +
|1µl
 +
|-
 +
|Primer 16
 +
|2,5µl
 +
|-
 +
|Primer 13k short
 +
|2,5µl
 +
|-
 +
|DMSO
 +
|1,5µl
 +
|-
 +
|template 7a(1:100)(850bp)
 +
|3µl -> 4ng
 +
|-
 +
|template 7b(new)(1:100)(402bp)
 +
|2µl -> 2ng
 +
|-
 +
|Phusion Hot Start II
 +
|0,5µl
 +
|}
 +
:->same with Primer 13l long<-
 +
 
 +
<font color="#009933">Overnight culture of PCR1 in cam- Vector</font>
 +
 
 +
new inoculation of PCR1 in cam, Colony 3 in 5ml LB+cam
 +
 
 +
[[Image:23 9 10 2apo.jpg|thumb|right|From left to right: PCR8k,PCR8l,ladder (5000,2000,850,400,100bp)
 +
]]
 +
 
 +
<font color="#009933">Transformation of pSB1C3, BB1, BB6</font>
 +
 
 +
New transformation of pSB1C3 from Spring distribution because the old colony was pink.
 +
 
 +
Transformation of BB1 and BB6.
 +
 
 +
 
 +
BB1 plated on amp-Agar, BB6 and pSB1C3 plated on cam-Agar
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/19 Transformation SSS|19 Transformation (Sina Science Services)]]
 +
 
 +
<font color="#009933">Agarose gelelectrophoresis of PCR8 (long and short primer 13)</font>
 +
 
 +
PCR8k=short primer 13, PCR8l=long primer 13
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
 +
 
 +
result:<font color="#CC33CC"> '''PCR 8k and 8l shows right band with ~1200bp'''</font>
 +
 
 +
<font color="#009933">PCR purification of PCR 8</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/12 Gel extraction or PCR Clean up| 12 Gel extraction or PCR Clean up (Promega kit)]]
 +
 
 +
result: ~70ng/µl; A260/A280=1.75
 +
 
== 9-24-2010 ==
== 9-24-2010 ==
-
text
+
<font color="#009933">New "over-day" culture of pSB1C3</font>
 +
 
 +
Inoculate one culture from the ligation yesterday in 4ml LB+cam
 +
 
 +
<font color="#009933">Ligation of BB7<sub>2</sub></font>
 +
 
 +
Mixture:
 +
 
 +
Inserts: each 5µl; ccdB vector: 1µl; T4 Ligase: 0.5µl; T4 Ligase Buffer: 2µl; H<sub>2</sub>O: 7µl
 +
 
 +
Incubation: 5h 18°C; Inhibition: 20 min 65°C
 +
 
 +
->Protocols: [[Team:LMU-Munich/Notebook/Protocols/22 Ligation|22 Ligation]], [[Team:LMU-Munich/Notebook/Protocols/13 3A Method for Biobrick assembly| 13 3A Method for Biobrick assembly]]
 +
 
 +
<font color="#009933">Colony PCRs of BB1 and BB6</font>
 +
 
 +
2 Colonys were picked from each plate -> 4 charges
 +
 
 +
Mixture for 4 charges with 5µl:
 +
 
 +
{|
 +
|PCR Mastermix 2x
 +
|10µl
 +
|-
 +
|Primer F
 +
|1.5µl
 +
|-
 +
|Primer R
 +
|1.5µl
 +
|-
 +
|H<sub>2</sub>O
 +
|7µl
 +
|-
 +
|sum
 +
|20µl
 +
|-
 +
|}
 +
 
 +
+ Colony
 +
 
 +
[[Image: apo-24.9.10.jpg|200px|thumb|right|from left to right: 1<sub>1</sub>, 1<sub>2</sub>, 6<sub>1</sub> and 6<sub>2</sub>]]
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]]
 +
 
 +
Annealing temperature: 53°C, standard protocol
 +
 
 +
<font color="#009933">Plasmid extraction of new PCR1 in cam vector</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]]
 +
 
 +
results:
 +
 
 +
{|
 +
| concentration
 +
| 82.5ng/µl
 +
|-
 +
| A260/A280
 +
| 1.737
 +
|}
 +
 
 +
 
 +
<font color="#009933">Agarose Gelelektrophoresis of 1<sub>1</sub>, 1<sub>2</sub>, 6<sub>1</sub> and 6<sub>2</sub></font>
 +
 
 +
 
 +
<font color="#009933">Plasmid extraction of pSB1C3 in cam vector</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]]
 +
 
 +
results:
 +
 
 +
{|
 +
| concentration
 +
| 22.5ng/µl
 +
|-
 +
| A260/A280
 +
| 1.5
 +
|}
 +
 
== 9-25-2010 ==
== 9-25-2010 ==
weekend
weekend
Line 1,393: Line 4,072:
== 9-27-2010 ==
== 9-27-2010 ==
-
text
+
 
 +
<font color="#009933">Colony PCR of BB7<sub>2</sub>, BB6 and BB1 plus a negativ charge as reference</font>
 +
 
 +
{|
 +
| PCR Mastermix
 +
| 17.5µl
 +
|-
 +
| Primer forward
 +
| 2.625µl
 +
|-
 +
| Primer revers
 +
| 2.7µl
 +
|-
 +
| H<sub>2</sub>O
 +
| 12.1µl
 +
|-
 +
| sum
 +
| 35µl = 5µl per charge
 +
|}
 +
 
 +
-> Protocol [[Team:LMU-Munich/Notebook/Protocols/24 Colony PCR|24 Colony PCR]]
 +
 
 +
 
 +
<font color="#009933">Ligation of BB10</font>
 +
 
 +
{|
 +
| CMV© E+S
 +
| 5µl
 +
|-
 +
| PCR 8l X+P
 +
| 5µl
 +
|-
 +
| ccdBamp
 +
| 1µl
 +
|-
 +
| T4 Ligase-Buffer
 +
| 2µl
 +
|-
 +
| T4 Ligase
 +
| 0.5µl
 +
|-
 +
| H<sub>2</sub>O
 +
| 6.5µl
 +
|-
 +
| sum
 +
| 20µl
 +
|}
 +
 
 +
-> Protocol [[Team:LMU-Munich/Notebook/Protocols/22 Ligation|22 Ligation]]
 +
 
 +
 
 +
<font color="#009933">Ligation of BB001(PCR1 in ©), BB017(PCR5 in ©), BB018(PCR6 in ©) and BB021(PCR9 in ©)</font>
 +
 
 +
{|
 +
| ccdB
 +
| 1µl
 +
|-
 +
| BB001/017/018/021
 +
| 5µl
 +
|-
 +
| T4 Ligase-Buffer
 +
| 2µl
 +
|-
 +
| T4 Ligase
 +
| 0.5µl
 +
|-
 +
| H<sub>2</sub>O
 +
| 11.5µl
 +
|-
 +
| sum
 +
| 20µl
 +
|}
 +
 
 +
-> Protocol [[Team:LMU-Munich/Notebook/Protocols/22 Ligation|22 Ligation]]
 +
 
 +
<font color="#009933">Restriction digestion of PCR3<sub>2</sub>, 8l, 10 and Primer 18+19 with E+P</font>
 +
 
 +
{| class="wikitable" border="1"
 +
|-
 +
!
 +
! template
 +
! H<sub>2</sub>O
 +
! BSA [1:10]
 +
! Buffer H
 +
! EcoRI
 +
! PstI
 +
|-
 +
| PCR3<sub>2</sub>
 +
| 10µl
 +
| 5µl
 +
| 2µl
 +
| 2µl
 +
| 0.5µl
 +
| 0.5µl
 +
|-
 +
| PCR8l
 +
| 10µl
 +
| 5µl
 +
| 2µl
 +
| 2µl
 +
| 0.5µl
 +
| 0.5µl
 +
|-
 +
| Primer 18+19
 +
| 2x7.5µl
 +
| --
 +
|2µl
 +
| 2µl
 +
| 0.5µl
 +
| 0.5µl
 +
|-
 +
| PCR10
 +
| 10µl
 +
| 5µl
 +
| 2µl
 +
| 2µl
 +
| 0.5µl
 +
| 0.5µl
 +
|}
 +
 
 +
-> Protocol  [[Team:LMU-Munich/Notebook/Protocols/5 Restriction digest|5 Restriction digestion]]
 +
 
== 9-28-2010 ==
== 9-28-2010 ==
-
text
+
<font color="#009933">Plating BB017, BB021, BB018, BB001, BB7<sub>2</sub>, BB10</font>
 +
 
 +
{| class="wikitable" border="1"
 +
|-
 +
| Amp
 +
| BB7<sub>2</sub>
 +
| BB10
 +
|
 +
|
 +
|-
 +
| Cam
 +
| BB001
 +
| BB017
 +
| BB018
 +
| BB021
 +
|}
 +
 
 +
<font color="#009933">Colony PCR  BB1, BB6, BB7<sub>2</sub> with DreamTaq (each 2 colonies)</font>
 +
 
 +
-> Protocol: [[Team:LMU-Munich/Notebook/Protocols/16 PCR with DreamTaq| 16 PCR with DreamTaq]]
 +
 
 +
PCR mixture for PCR7a
 +
::{|
 +
|-
 +
|template
 +
|pick up colony from plate
 +
|-
 +
|primers*2
 +
|0.25µl*2
 +
|-
 +
|dNTPs
 +
|0.5µl
 +
|-
 +
|DreamTaq
 +
|0.05µl
 +
|-
 +
|10xbuffer
 +
|0.5µl
 +
|-
 +
|H<sub>2</sub>O
 +
|3.45µl
 +
|-
 +
|sum
 +
|5µl
 +
|}
 +
 
 +
Primers for colony PCR: BBseqF, BBseqR
 +
 
 +
Annealing temperature: 53°C
 +
 
 +
<font color="#009933">Ligation of BB2, BB019, BB020, BB016, BB022</font>
 +
 
 +
{| class="wikitable" border="1"
 +
|-
 +
!
 +
! BB2
 +
! BB019
 +
! BB020
 +
! BB016
 +
! BB022
 +
! Volume
 +
|-
 +
| PCR
 +
| 5<sub>2</sub>
 +
| 8
 +
|
 +
| 3<sub>2</sub>
 +
| 10
 +
| each 5µl
 +
|-
 +
| PCR
 +
| 6
 +
|
 +
|
 +
|
 +
|
 +
| each 5µl
 +
|-
 +
| Primer
 +
|
 +
|
 +
| 18+19
 +
|
 +
|
 +
| each 5µl
 +
|-
 +
| ccdBamp
 +
| 1µl
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|-
 +
| ccdBcam
 +
|
 +
| 1µl
 +
| 1µl
 +
| 1µl
 +
| 1µl
 +
|
 +
|-
 +
| T4 Ligase-Buffer
 +
| 2µl
 +
| 2µl
 +
| 2µl
 +
| 2µl
 +
| 2µl
 +
|
 +
|-
 +
| T4 Ligase
 +
| 0.5µl
 +
| 0.5µl
 +
| 0.5µl
 +
| 0.5µl
 +
| 0.5µl
 +
|
 +
|-
 +
| H<sub>2</sub>O
 +
| 6.5µl
 +
| 11.4µl
 +
| 11.4µl
 +
| 11.4µl
 +
| 11.4µl
 +
|
 +
|-
 +
| sum
 +
| 20µl
 +
| 20µl
 +
| 20µl
 +
| 20µl
 +
| 20µl
 +
|
 +
|}
 +
 
== 9-29-2010 ==
== 9-29-2010 ==
-
text
+
plates BB7<sub>2</sub>, BB017, BB10, BB021, BB001, BB018: nothing grown -> thrown into trash
 +
 
 +
<font color="#009933">colony-PCR of BB2/8, BB019, BB016, BB020, BB022 with Taq</font>
 +
 
 +
->protocol: [[Team:LMU-Munich/Notebook/Protocols/25 PCR with Taq|PCR with Taq]]
 +
 
 +
{|
 +
|-
 +
|H<sub>2</sub>O
 +
|6,7µl
 +
|-
 +
|Buffer 10x
 +
|1µl
 +
|-
 +
|MgCl<sub>2</sub>
 +
|1µl
 +
|-
 +
|dNTPs
 +
|0,2µl
 +
|-
 +
|Primer forw. seq f
 +
|0,5µl
 +
|-
 +
|Primer rev. seq r
 +
|0,5µl
 +
|-
 +
|Taq
 +
|0,1µl
 +
|}
 +
 
 +
template: pick colony
 +
 
 +
sum: 10µl; we made 100µl ->10 charges (2 colonies of each plate)
 +
 
 +
tube numbers:
 +
{| class="wikitable" border="1"
 +
|-
 +
|1
 +
|2
 +
|3
 +
|4
 +
|5
 +
|6
 +
|7
 +
|8
 +
|9
 +
|10
 +
|-
 +
|BB2/BB8 - 1
 +
|BB2/BB8 - 2
 +
|BB016 - 1
 +
|BB016 - 2
 +
|BB019 - 1
 +
|BB019 - 2
 +
|BB020 - 1
 +
|BB020 - 2
 +
|BB022 - 1
 +
|BB022 - 2
 +
|}
 +
 
 +
PCR-program: taq53
 +
 
 +
agarose-gelelektrophoresis -> no bands visible
 +
 
 +
<font color="#009933">new plates made of BB1, BB4, BB6, BB7</font>
 +
 
 +
<font color="#009933">Ligations BB7<sub>2</sub>, BB017 (mit PCR5<sub>1</sub>), BB10, BB021, BB001, BB018</font>
 +
 
 +
{| class="wikitable" border="1"
 +
|-
 +
|
 +
|PCR (5 µl each)
 +
|PCR (5µl each)
 +
|ccdB(amp)
 +
|ccdB©
 +
|Buffer
 +
|T4 Ligase
 +
|H<sub>2</sub>O
 +
|-
 +
|BB7<sub>2</sub>O
 +
|1
 +
|3<sub>2</sub>
 +
|1µl
 +
|
 +
|2µl
 +
|0,5µl
 +
|6,5µl
 +
|-
 +
|BB017
 +
|5<sub>1</sub>
 +
|
 +
|
 +
|1µl
 +
|2µl
 +
|0,5µl
 +
|11,5µl
 +
|-
 +
|BB10
 +
|CMV©
 +
|PCR8
 +
|1µl
 +
|
 +
|2µl
 +
|0,5µl
 +
|6,5µl
 +
|-
 +
|BB021
 +
|9
 +
|
 +
|
 +
|1µl
 +
|2µl
 +
|0,5µl
 +
|11,5µl
 +
|-
 +
|BB001
 +
|1
 +
|
 +
|
 +
|1µl
 +
|2µl
 +
|0,5µl
 +
|11,5µl
 +
|-
 +
|BB018
 +
|6
 +
|
 +
|
 +
|1µl
 +
|2µl
 +
|0,5µl
 +
|11,5µl
 +
|}
 +
 
 +
<font color="#009933">restriction degestions</font>
 +
 
 +
of PCR6, PCR5<sub>2</sub>, PCR3<sub>2</sub>, PCR1, ccdB(amp), ccdB(cam)
 +
 
 +
{| class="wikitable" border="1"
 +
|-
 +
|template
 +
|digestion with
 +
|volume
 +
|-
 +
|PCR 6
 +
|Xba1 & Pst1
 +
|I. 10µl  II. 3µl
 +
|-
 +
|PCR5<sub>2</sub>
 +
|EcoR1 & Pst1
 +
|20µl
 +
|-
 +
|PCR3<sub>2</sub>
 +
|Xba1 & Pst1
 +
|10µl
 +
|-
 +
|PCR1
 +
|EcoR1 & Spe1
 +
|2µl
 +
|-
 +
|ccdB(amp)
 +
|EcoR1 & Pst1
 +
|20µl
 +
|-
 +
|cddB(cam)
 +
|EcoR1 & Pst1
 +
|20µl
 +
|}
 +
->protocol: [[Team:LMU-Munich/Notebook/Protocols/5 Restriction digest|5 Restriction digestion]]
 +
 
 +
 
 +
<font color="#009933">colony-PCR of BB4/c3, BB6/c7, BB7<sub>2</sub>/c5, BB1/c5, (BB11 as control) with Taq</font>
 +
 
 +
->protocol: [[Team:LMU-Munich/Notebook/Protocols/25 PCR with Taq|PCR with Taq]]
 +
 
 +
Mastermix:
 +
 
 +
{|
 +
|-
 +
|H<sub>2</sub>O
 +
|32µl
 +
|-
 +
|Buffer 10x
 +
|5µl
 +
|-
 +
|MgCl<sub>2</sub>
 +
|5µl
 +
|-
 +
|dNTPs
 +
|1µl
 +
|-
 +
|Primer forw. seq f
 +
|2,5µl
 +
|-
 +
|Primer rev. seq r
 +
|2,5µl
 +
|-
 +
|Taq
 +
|0,5µl
 +
|-
 +
|DMSO
 +
|1,5µl
 +
|-
 +
|sum
 +
|50µl
 +
|-
 +
|}
 +
 
 +
->10µl for each charge
 +
 
 +
template: pick colony; BB11: 2ng
 +
 
 +
tube numbers:
 +
{| class="wikitable" border="1"
 +
|-
 +
|1
 +
|2
 +
|3
 +
|4
 +
|5
 +
|-
 +
|BB4-c3
 +
|BB6-c7
 +
|BB7<sub>2</sub>-c5
 +
|BB1-c5
 +
|BB11
 +
|-
 +
|}
 +
 
 +
PCR program:T<sub>A</sub> = 53°C; elongation time=1min
 +
 
== 9-30-2010 ==
== 9-30-2010 ==
-
text
+
<font color="#009933">Colony PCR of BB2/BB8, BB6 K7&K8 and test PCR of BB11 with new dilutions and primers</font>
 +
 
 +
{|
 +
| Colony/BB11
 +
| 2.5µl
 +
|-
 +
| H<sub>2</sub>O
 +
| 32µl
 +
|-
 +
| dNTP
 +
| 1µl
 +
|-
 +
| Primer F
 +
| 2.5µl
 +
|-
 +
| Primer R
 +
| 2.5µl
 +
|-
 +
| MgCl<sub>2</sub>
 +
| 5µl
 +
|-
 +
| DMSO
 +
| 1.5µl
 +
|-
 +
| Buffer 10x (with KCl)
 +
| 5µl
 +
|-
 +
| Taq Polymerase
 +
| 0.5µl
 +
|-
 +
| sum
 +
| 56µl
 +
|}
 +
 
 +
-> Protocol [[Team:LMU-Munich/Notebook/Protocols/24 Colony PCR|24 Colony PCR]]
 +
 
 +
<font color="#009933">PCR purification of the digestions of 6I, 6II, 5<sub>2</sub>, 3<sub>2</sub>, 1</font>
 +
 
 +
{|
 +
|
 +
| concentration [ng/µl]
 +
| A260/A280
 +
|-
 +
| 6II
 +
| 32.5
 +
| 1.444
 +
|-
 +
| ccdBamp
 +
| 0.0
 +
| ---
 +
|-
 +
| ccdBcam
 +
| 5.0
 +
| 1.000
 +
|-
 +
| 6I
 +
| 0.0
 +
| ---
 +
|-
 +
| 5<sub>2</sub>
 +
| 20.0
 +
| 1.143
 +
|-
 +
| 3<sub>2</sub>
 +
| 27.5
 +
| 1.57
 +
|-
 +
| 1
 +
| 25
 +
| 1.667
 +
|}
 +
 
 +
-> Protocol [[Team:LMU-Munich/Notebook/Protocols/12 Gel extraction or PCR Clean up| 12 Gel extraction or PCR Clean up (Promega kit)]]
 +
 
 +
<font color="#009933">Transformation of BB018, BB7<sub>2</sub>, BB001, BB017, BB10 and BB021</font>
 +
 
 +
-> Protocol [[Team:LMU-Munich/Notebook/Protocols/19 Transformation SSS|19 Transformation SSS]]
 +
 
 +
<font color="#009933">Plasmidextraction of the following overnight cultures: BB020(K1), BB018(K5), BB6(K5), BB022(K1), BB016(K1), BB019(K1), BB2(K1) and BB018(K3)</font>
 +
 
 +
{|
 +
|
 +
| concentration [ng/µl]
 +
| A260/A280
 +
|-
 +
| BB020(K1)
 +
| 163
 +
| 1.711
 +
|-
 +
| BB018(K5)
 +
| 92.5
 +
| 1.85
 +
|-
 +
| BB6(K5)
 +
| 120
 +
| 1.778
 +
|-
 +
| BB022(K1)
 +
| 105
 +
| 1.75
 +
|-
 +
| BB016(K1)
 +
| 85
 +
| 1.889
 +
|-
 +
| BB019(K1)
 +
| 105
 +
| 1.909
 +
|-
 +
| BB2(K1)
 +
| 190
 +
| 1.81
 +
|-
 +
| BB018
 +
| 110
 +
| 1.833
 +
|}
 +
 
 +
-> Protocol [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]]
 +
 
== 10-01-2010 ==
== 10-01-2010 ==
-
text
+
<font color="#009933">new test PCR of BB5 and BB11 with 20µl and 50µl volume</font>
 +
 
 +
Master Mix
 +
{|
 +
| H<sub>2</sub>O
 +
| 67µl
 +
|-
 +
| dNTP
 +
| 2µl
 +
|-
 +
| Primer F
 +
| 5µl
 +
|-
 +
| Primer R
 +
| 5µl
 +
|-
 +
| MgCl<sub>2</sub>
 +
| 10µl
 +
|-
 +
| Buffer 10x (with KCl)
 +
| 10µl
 +
|-
 +
| Taq Polymerase
 +
| 1µl
 +
|-
 +
| sum
 +
| 100µl
 +
|}
 +
 
 +
template: BB5/50µl: 5ng, 20µl: 1.5ng; BB11/20µl: 3ng
 +
 
 +
->protocol: [[Team:LMU-Munich/Notebook/Protocols/25 PCR with Taq|PCR with Taq]]
 +
 
 +
[[Image:01.10.10. apo.jpg|01.10.10. Gel photo]]
 +
 
 +
from left to right: ladder, BB5/50µl, BB5/20µl, BB11/20µl. BB5/50µl and BB11/20µl show the right band.
 +
 
== 10-02-2010 ==
== 10-02-2010 ==
weekend
weekend
== 10-03-2010 ==
== 10-03-2010 ==
weekend
weekend
 +
 +
== 10-04-2010 ==
 +
sequencing
 +
 +
cycle, clean and run
 +
 +
{| class="wikitable" border="1"
 +
|-
 +
!
 +
! CMV (c)
 +
! PCR 1 (c)
 +
! BB4 <sub>7</sub>
 +
! BB4 <sub>8</sub>
 +
! BB018K5
 +
! BB018K3
 +
! BB6K5
 +
! BB2K1
 +
|-
 +
| template
 +
| 1µl
 +
| 2µl
 +
| 3µl
 +
| 3,8µl
 +
| 2µl
 +
| 2µl
 +
| 2µl
 +
| 1µl
 +
|-
 +
| primer (1:100)
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
|-
 +
| 10mM TrispH 8,5
 +
| 2,8 µl
 +
| 1,8 µl
 +
| 0,8 µl
 +
| 0 µl
 +
| 1,8 µl
 +
| 1,8 µl
 +
| 1,8 µl
 +
| 1,8 µl
 +
|-
 +
|name of tube
 +
| 028 FIR
 +
| 001 F/R
 +
| 4-7 F/R
 +
| 4-8 F/R
 +
| 018-5 F/R
 +
| 018-3 F/R
 +
| 6-5 F/R
 +
| 2-1 F/R
 +
|}
 +
 +
 +
PCR
 +
 +
{| class="wikitable" border="1"
 +
|-
 +
!
 +
! 019
 +
! 022
 +
! 016
 +
! 020
 +
|-
 +
| template (1:100 = 2ng)
 +
| 2µl
 +
| 2µl
 +
| 2,5µl
 +
| 1,5µl
 +
|-
 +
| Primer F
 +
| 1µl
 +
| 1µl
 +
| 1µl
 +
| 1µl
 +
|-
 +
| Primer R
 +
| 1µl
 +
| 1µl
 +
| 1µl
 +
| 1µl
 +
|-
 +
| dNTPs
 +
| 0,4µl
 +
| 0,4µl
 +
| 0,4µl
 +
| 0,4µl
 +
|-
 +
| Taq
 +
| 0,2µl
 +
| 0,2µl
 +
| 0,2µl
 +
| 0,2µl
 +
|-
 +
| Buffer
 +
| 2µl
 +
| 2µl
 +
| 2µl
 +
| 2µl
 +
|-
 +
|MgCl<sub>2</sub>
 +
| 2µl
 +
| 2µl
 +
| 2µl
 +
| 2µl
 +
|-
 +
|H<sub>2</sub>O
 +
| 11,4µl
 +
| 11,4µl
 +
| 10,9µl
 +
| 11,
 +
|-
 +
|colour of tube
 +
| orange
 +
| rose
 +
| green
 +
| blue
 +
|}
 +
 +
== 10-05-2010 ==
 +
<font color="#009933">Colony PCRs of BB6, BB4, BB021, BB022, CMV©, BB001, BB016, BB1, BB2<sub>5<sub>2</sub></sub>, BB7<sub>2</sub>, BB017, BB018, BB019, BB020</font>
 +
 +
<font color="#009933">agarose gel electrophoresis</font>
 +
 +
schedule of the agarose gel electrophoresis:
 +
 +
{| class="wikitable" border="1"
 +
| gel 1
 +
| BB6K9
 +
| BB6K10
 +
| BB6K11
 +
| BBK12
 +
| BB6K13
 +
| BB4K5
 +
| ladder
 +
| BB4K6
 +
| BB4K7
 +
|-
 +
|
 +
| BB021K14
 +
| BB021K13
 +
| BB021K12
 +
| BB021K11
 +
| BB022K18
 +
| BB022K19
 +
| ladder
 +
| BB022K17
 +
| BB022K16
 +
|-
 +
| gel 2
 +
| BB022K12
 +
| BB022K11
 +
| CMV©K4
 +
| CMV©K5
 +
| BB001K5
 +
| BB001K6
 +
| BB001K7
 +
| BB001K8
 +
| ladder
 +
|-
 +
|
 +
| BB016K17
 +
| BB1K16
 +
| BB1K15
 +
| BB1K8
 +
| BB1K7
 +
| BB2<sub>5<sub>2</sub></sub>K1
 +
| BB2<sub>5<sub>2</sub></sub>K2
 +
| BB2<sub>5<sub>2</sub></sub>K3
 +
| ladder
 +
|-
 +
| gel 3
 +
| BB017K14
 +
| BB017K13
 +
| BB017K12
 +
| BB017K11
 +
| BB018K17
 +
| BB018K16
 +
| BB018K15
 +
| BB018K14
 +
| ladder
 +
|-
 +
|
 +
| BB019K6
 +
| BB019K5
 +
| BB019K4
 +
| ladder
 +
| BB019K3
 +
| BB020K7
 +
| BB020K8
 +
| BB020K6
 +
| BB020K5
 +
|}
 +
 +
{| class="wikitable" border="1"
 +
| gel 1
 +
| BB4K8
 +
| BB4K9
 +
| BB4K10
 +
| BB021K10
 +
| BB021K15
 +
|
 +
|
 +
|
 +
|
 +
|-
 +
|
 +
| BB022K15
 +
| BB022K14
 +
| BB022K13
 +
| ---
 +
| ---
 +
|
 +
|
 +
|
 +
|
 +
|-
 +
| gel 2
 +
| BB001K9
 +
| BB001K10
 +
| BB016K11
 +
| BB016K12
 +
| BB016K13
 +
| BB016K14
 +
| BB016K15
 +
| BB016K16
 +
| ---
 +
|-
 +
|
 +
| ---
 +
| BB2<sub>5<sub>2</sub></sub>K4
 +
| BB2<sub>5<sub>2</sub></sub>K5
 +
| BB7<sub>2</sub>K12
 +
| BB7<sub>2</sub>K11
 +
| BB7<sub>2</sub>K10
 +
| BB7<sub>2</sub>K9
 +
| BB017K16
 +
| BB017K15
 +
|-
 +
|gel 3
 +
| BB018K13
 +
| BB018K12
 +
| BB018K11
 +
| BB018K10
 +
| BB018K9
 +
|
 +
|
 +
|
 +
|
 +
|-
 +
|
 +
| BB020K4
 +
| BB020K3
 +
| ---
 +
| ---
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|}
 +
 +
Gelbilder und Auswertung einfügen
 +
 +
[[Image:5.10.10apo1,2,3.jpg|400px|9 15 10 Gelphoto]]
 +
 +
 +
 +
<font color="#009933">overnight cultures of BB6K3, BB4K10, CMV©K4, BB001K5, BB001K9, BB016K11, BB1K8, BB2<sub>5<sub>2</sub></sub>K3, BB7<sub>2</sub>, BB017K14, BB018K10, BB019K6</font>
 +
 +
== 10-06-2010 ==
 +
 +
<font color="#009933">Plasmidextraction of the following overnight cultures: BB6(K13), BB4(K13), CMVc (K4), BB001(K5), BB001(K9), BB016(K11), BB1(K8), BB2(K3), BB7(K10), BB017(K14), BB018(K10) and BB019(K6) </font>
 +
 +
{|
 +
|
 +
| concentration [ng/µl]
 +
| A260/A280
 +
|-
 +
| BB6(K13)
 +
| 265
 +
| 1.8
 +
|-
 +
| BB4(K10)
 +
| 40
 +
| 2.0
 +
|-
 +
| CMVc(K4)
 +
| 97.5
 +
| 1.8
 +
|-
 +
| BB001(K5)
 +
| 30
 +
| 1.7
 +
|-
 +
| BB001(K9)
 +
| 17.5
 +
| 1.7
 +
|-
 +
| BB016(K11)
 +
| 45
 +
| 1.7
 +
|-
 +
| BB1(K8)
 +
| 57.5
 +
| 1.9
 +
|-
 +
| BB2(K3)
 +
| 60
 +
| 1.8
 +
|-
 +
| BB7(K10)
 +
| 30
 +
| 1.7
 +
|-
 +
| BB017(K14)
 +
| 12.5
 +
| 1.5
 +
|-
 +
| BB018(K10)
 +
| 35
 +
| 1.8
 +
|-
 +
|BB019(K6)
 +
| 37.5
 +
| 1.5
 +
|-
 +
 +
|}
 +
 +
-> Protocol [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]]
 +
 +
== 10-07-2010 ==
 +
 +
<font color="#009933"> colony PCR of BB021, BB022 with Taq (53°C) </font>
 +
 +
 +
 +
{|
 +
| Primer fw
 +
| 0.5 µl
 +
|-
 +
| Primer rev
 +
| 0.5 µl
 +
|-
 +
| dNTPs
 +
| 0.2 µl
 +
|-
 +
| Taq
 +
| 0.1 µl
 +
|-
 +
| 10xbuffer
 +
| 1 µl
 +
|-
 +
| MgCl<sub>2</sub>
 +
| 1 µl
 +
|-
 +
| H<sub>2</sub>O
 +
| 6.7 µl
 +
|-
 +
 +
|}
 +
 +
== 10-08-2010 ==
 +
 +
 +
<font color="#009933">
 +
plasmide extractions from 10-06-2010 were put in a lyophilizer and redissolved in less water (10-20 µl)
 +
</font>
 +
{|
 +
|
 +
| concentration [ng/µl]
 +
| A260/A280
 +
|-
 +
| BB4(K10)
 +
|    258
 +
|    1,981
 +
|-
 +
| BB001(K5)
 +
|    325
 +
|    1.94
 +
|-
 +
| BB001(K9)
 +
|    205
 +
|    1.907
 +
|-
 +
| BB7(K10)
 +
|    170
 +
|    1.744
 +
|-
 +
| BB017(K14)
 +
|    215
 +
|    1.955
 +
|-
 +
| BB018(K10)
 +
|    500
 +
|    1.88
 +
|-
 +
|BB019(K6)
 +
|    293
 +
|    1.95
 +
|-
 +
 +
|}
 +
 +
== 10-09-2010 ==
 +
<font color="#009933">Plasmid extraction BB018K5, BB018K6, BB021K17 and BB021K24</font color>
 +
 +
concentration of these samples to be measured on Monday
 +
 +
-> Protocol [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]]
 +
 +
== 10-10-2010 ==
 +
weekend
 +
 +
== 10-11-2010 ==
 +
 +
 +
<font color="#009933">Plasmid extraction BB018K5, BB018K6, BB021K24 and BB021K17 from 10-09-2010
 +
</font>
 +
 +
 +
{|
 +
|
 +
| concentration [ng/µl]
 +
| A260/A280
 +
|-
 +
| BB018K5
 +
|    55
 +
|    1.571
 +
|-
 +
| BB018K6
 +
|    92,5
 +
|    1.762
 +
|-
 +
| BB021K24
 +
|    27.5
 +
|    1.833
 +
|-
 +
| BB021K17
 +
|    37.5
 +
|    1.508
 +
|-
 +
 +
|}
 +
 +
 +
<font color="#009933">Colony PCR BB01, BB018, BB6, BB252</font>
 +
 +
{|
 +
| stripe 1
 +
| BB001K5
 +
| K6
 +
| K7
 +
| K8
 +
| K9
 +
| K9
 +
| K10
 +
| BB252 K2
 +
|-
 +
| stripe 2
 +
| BB252 K3
 +
| K4
 +
| K5
 +
| BB6 K9
 +
| K10
 +
| K11
 +
| K12
 +
| K13
 +
|-
 +
| stripe 3
 +
| BB018 K9
 +
| K10
 +
| K11
 +
| K12
 +
| K13
 +
| K14
 +
| K15
 +
| K16
 +
|-
 +
|single sample
 +
|BB018 K17
 +
|-
 +
 +
|}
 +
 +
 +
gel picture colony PCR
 +
 +
foto einfügen!
 +
 +
<font color="#009933">Sequencing 8,9 and 10 (cycle, clean and run)</font>
 +
 +
{| class="wikitable" border="1"
 +
|-
 +
!
 +
! BB001K9 for
 +
! BB001K9 rev
 +
! BB4K16 for
 +
! BB4K16 rev
 +
! BB001K5 for
 +
! BB001K5 rev
 +
! BB2<sub>5<sub>2</sub></sub>K3
 +
! BB2<sub>5<sub>2</sub></sub>K3
 +
|-
 +
| template
 +
| 1µl
 +
| 1µl
 +
| 1µl
 +
| 1µl
 +
| 0,5µl
 +
| 0,5µl
 +
| 3,8µl
 +
| 3,8µl
 +
|-
 +
| primer (1:100)
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
|-
 +
| 10mM TrispH 8,5
 +
| 2,8 µl
 +
| 2,8 µl
 +
| 2,8 µl
 +
| 2,8 µl
 +
| 3,3 µl
 +
| 3,3 µl
 +
| 0 µl
 +
| 0 µl
 +
|-
 +
|name of tube
 +
| 31-F
 +
| 31-R
 +
| 44-F
 +
| 44-R
 +
| 24-F
 +
| 24-R
 +
| 32-F
 +
| 32-R
 +
|}
 +
 +
 +
{| class="wikitable" border="1"
 +
|-
 +
!
 +
! BB7<sub>2</sub>K10 for
 +
! BB7<sub>2</sub>K10 rev
 +
! BB017K14 for
 +
! BB017K14 rev
 +
! BB018K10 for
 +
! BB018K10 rev
 +
! BB019K6 for
 +
! BB019K6 rev
 +
|-
 +
| template
 +
| 1,5µl
 +
| 1,5µl
 +
| 1µl
 +
| 1µl
 +
| 0,5µl
 +
| 0,5µl
 +
| 0,8µl
 +
| 0,8µl
 +
|-
 +
| primer (1:100)
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
|-
 +
| 10mM TrispH 8,5
 +
| 2,3 µl
 +
| 2,3 µl
 +
| 2,8 µl
 +
| 2,8 µl
 +
| 3,3 µl
 +
| 3,3 µl
 +
| 3 µl
 +
| 3 µl
 +
|-
 +
|name of tube
 +
| 14-F
 +
| 14-R
 +
| 21-F
 +
| 21-R
 +
| 13-F
 +
| 13-R
 +
| 34-F
 +
| 34-R
 +
|}
 +
 +
 +
{| class="wikitable" border="1"
 +
|-
 +
!
 +
! CMV for
 +
! CMV rev
 +
! BB018K5 for
 +
! BB018K5 rev
 +
! BB018K6 for
 +
! BB018K6 rev
 +
! BB021K17
 +
! BB021K17
 +
|-
 +
| template
 +
| 2µl
 +
| 2µl
 +
| 3,8µl
 +
| 3,8µl
 +
| 2µl
 +
| 2µl
 +
| 3,8µl
 +
| 3,8µl
 +
|-
 +
| primer (1:100)
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
| 3,2 µl
 +
|-
 +
| 10mM TrispH 8,5
 +
| 1,8 µl
 +
| 1,8 µl
 +
| 0 µl
 +
| 0 µl
 +
| 1,8 µl
 +
| 1,8 µl
 +
| 0 µl
 +
| 0 µl
 +
|-
 +
|name of tube
 +
| CMV-F
 +
| CMV-R
 +
| 185-F
 +
| 185-R
 +
| 186-F
 +
| 186-R
 +
| 2117-F
 +
| 2117-R
 +
|}
 +
 +
 +
<font color="#009933">ligation BB020 BB022, Bb6 and BB5<sub>2</sub></font color>
 +
 +
-> Protocol [[Team:LMU-Munich/Notebook/Protocols/19 Transformation SSS|19 Transformation SSS]]
 +
 +
{| class="wikitable" border="1"
 +
|-
 +
!
 +
! BB020
 +
! BB022
 +
! BB6
 +
! BB5<sub>2</sub>
 +
|-
 +
| insert (5µl)
 +
| 18+19
 +
| PCR 10
 +
| CMV
 +
| PCR5<sub<2</sub>
 +
|-
 +
| insert (5µl)
 +
| -
 +
| -
 +
| BB5
 +
| PCR6
 +
|-
 +
| ccdB (1µl)
 +
| c
 +
| c
 +
| c
 +
| a
 +
|-
 +
| buffer
 +
| 2µl
 +
| 2µl
 +
| 2µl
 +
| 2µl
 +
|-
 +
| T4-ligase
 +
| 0,5µl
 +
| 0,5µl
 +
| 0,5µl
 +
| 0,5µl
 +
|-
 +
| H<sub>2</sub>O
 +
| 11,5µl
 +
| 11,5µl
 +
| 6,5µl
 +
| 6,5µl
 +
|}
 +
 +
 +
 +
<font color="#009933">PCR to test self-made taq</font color>
 +
 +
 +
{|
 +
| template eGFP
 +
| 1µl
 +
|-
 +
| Primer 20,21
 +
| each 1µl
 +
|-
 +
| buffer (10x)
 +
| 2µl
 +
|-
 +
| MgCl
 +
| 2µl
 +
|-
 +
| dNTPs
 +
| 0,2µl
 +
|-
 +
| taq
 +
| 0,3µl to 0,7µl (0,1µl steps)
 +
|-
 +
| H<sub>2</sub>O
 +
| fill to 20 µl   
 +
|-
 +
 +
|}
 +
 +
anealing temperature 58°C, elongation time 30''
 +
{{:Team:LMU-Munich/Templates/Page Footer}}
{{:Team:LMU-Munich/Templates/Page Footer}}

Latest revision as of 18:04, 27 October 2010


ApoControl Notebook

What we did

Short description of our work, our results and our supporters


The creation of certain constructs was necessary for our two systems for cell selection by means of apoptosis: “Cut’N’Survive” and “Jump-Or-Die”. We searched for sources of the DNA sequences we needed and found several supporters which are listed below.

Most genes and promoters were amplificated via PCR with overhang-primers with the BioBrick prefix or suffix. If the sequence contained a EcoR1-, Pst1-, Xba1-, Spe1- or Not1- restriction site, we used mutagenesis primers and fusioned both DNA parts by fusion PCR. All PCRs worked out, even the fusion PCRs.

The length of the PCR products were tested by agarose gel electrophoresis. We tried to sequence our PCR products, but obtained poor results and resorted to sequencing the plasmids.

In parallel, we made competent cells and multiplied ccdB (death gene)-vectors with different antibiotic resistances. All components were digested with the appropriate restriction enzymes. The samples were cleaned with a PCR clean up kit or dephosphorylated to reduce false ligations.

We ligated our constructs and several interim stages with the 3A-assembly according to our schedule. The ligations were transformed to E.coli DH5α strains and selected by antibiotics. Afterwards, some colonies were picked and we tested the insertion of the construct by colony PCR.

If the colony PCR resulted in bands of the right size, we extracted the plasmids from overnight cultures and sequenced the samples with forward and reverse BioBrick primers.

Unfortunately, not all BioBricks were cloned succesfully. However, we were able to produce 4 BioBricks, one of which represents a full construct while the other three are intermediates. The system wasn't completed on time, so we weren´t able to test them in eukarytic cell lines.


The protocols we used are listed here: Protocols

These Biobricks we submitted:

  • BBa_K368004: attP+eGFP+SV40PA
  • BBa_K368011: eGFP+SV40PA
  • BBa_K368016: TEVrecognition site+N-degron+SF3b155
  • BBa_K368019: TEV-Protease+p14*+TEVrecognition site

Sources, helpers and supporters:

  • Prof. Dr. Angelika Böttger :
    • prevTRE (tet-on CMV promoter; inducible by doxycycline in special cell lines)
    • supported the construction ideas and would have given us the cells and mediums we would have needed
    • SV40PA (Polyadenylation site): gave us a vector containing it
    • Human Bak: her assistant Erika Clement gave us appropriate cDNA

Contents>

Week Days
Monday Tuesday Wednesday Thursday Friday Saturday Sunday
31 8-02-2010 8-03-2010 8-04-2010 8-05-2010 8-06-2010 8-07-2010 8-08-2010
32 8-09-2010 8-10-2010 8-11-2010 8-12-2010 8-13-2010 8-14-2010 8-15-2010
33 8-16-2010 8-17-2010 8-18-2010 8-19-2010 8-20-2010 8-21-2010 8-22-2010
34 8-23-2010 8-24-2010 8-25-2010 8-26-2010 8-27-2010 8-28-2010 8-29-2010
35 8-30-2010 8-31-2010 9-01-2010 9-02-2010 9-03-2010 9-04-2010 9-05-2010
36 9-06-2010 9-07-2010 9-08-2010 9-09-2010 9-10-2010 9-11-2010 9-12-2010
37 9-13-2010 9-14-2010 9-15-2010 9-16-2010 9-17-2010 9-18-2010 9-19-2010
38 9-20-2010 9-21-2010 9-22-2010 9-23-2010 9-24-2010 9-25-2010 9-26-2010
39 9-27-2010 9-28-2010 9-29-2010 9-30-2010 10-01-2010 10-02-2010 10-03-2010
40 10-04-2010 10-05-2010 10-06-2010 10-07-2010 10-08-2010 10-09-2010 10-10-2010
41 10-11-2010 10-12-2010 10-13-2010 10-14-2010 10-15-2010 10-16-2010 10-17-2010
42 10-18-2010 10-19-2010 10-20-2010 10-21-2010 10-22-2010 10-23-2010 10-24-2010
43 10-25-2010 10-26-2010 10-27-2010 10-28-2010 10-29-2010 10-30-2010 10-31-2010

8-10-2010

Transforming competent cells

- eGFP Biobrick: BBa_I714891 SDY_eGFP (Kanamycin)

- TEV recogn N Degron SF3 = pDS7 (Ampicillin)

- TEV p14 recogn = 190-6 (Ampicillin)

-> Protocol: (3 Transformation)

- We added 2 µl DNA

- We plated out 200 µl


Plasmid Isolation

- CMV-Promoter Biobrick: BBa_J52034

-> Protocol:(4 Plasmid extraction from cells)

- Prepared overnight culture, measured concentration of DNA

-> Poor results -> thrown away

8-11-2010

New Plasmid Extraction

- CMV-Promoter Biobrick: BBa_J52034

-> Protocol: (4 Plasmid extraction from cells)

- Plasmid concentration: 143ng/µl


Prepared overnight culture of eGFP BBa_I714891

- 3 ml LB-Media + 4 µl Kanamycin

- Inoculated with 1 colony of BBa_I714891 -> 37°C


Prepared overnight culture of 190-6 and pDS7 and eGFP (BBa_I714891) in falcons

- for 190-6 and pDS7: 10µl Ampicillin + 10 ml LB-Media + colony of plate

- for eGFP: 13,3 µl Kanamycin + 10 ml LB-Media + 1 colony of plate


Restriction digestion of CMV-Promoter BBa_J52034 with EcoRI and PstI

H2Oddest, sterile 10,3 µl
RE10 + Buffer H 2,0 µl
acetylated BSA (18ng/µl) 0,2 µl
DNA (0,143µg/µl) 6,0 µl

-> mixed

- plus: EcoRI (10µg/µl): 0,5 µl resp. PstI (10µg/µl): 0,5 µl

- incubated at room temperature from 12:10 to 15:00, 1 hour at 37°C, 2 hours at 60°C

- frozen at -20°C


Prepare new/fresh overnight culture of CMV-Promoter Biobrick: BBa_J52034

- 1 ml of "old" culture + 3 ml LB-Media + 4 µl Kanamycin -> 37°C

8-12-2010

Plasmid Extraction of pDS7, eGFP, 190-6

-> Protocol: (4 Plasmid extraktion from cells)

- pDS7 (458ng/µl), eGFP (55ng/µl), 190-6 (193ng/µl)

Restriction digest of pDS7, eGFP, 190-6

- with EcoRI and PstI in buffer H (for testing DNA is correct)

-> Protocol: (5 Restriction digest)

- 10µg DNA: pDS7 (2µl), eGFP (15µl), 190-6 (10µl)

Plate colonies for plasmid extraction

- CMV (Kanamycin), eGFP (Kanamycin), pDS7 (Ampicillin), 190-6 (Ampicillin))

- PhiC31o plated on Ampicillin-Agar, stored at 37°C

50% Glycerol made

- for PhiC31o glycerol stock (produced later)

8-13-2010

Gelfoto from the EcoR1 and Pst1 Restrictiondigest of 190-6, eGFP, pDS7 and CMV

Inoculate CMV into LB medium with ampicillin

- CMV (BBa_J52034) from 10.8.2010 inoculated into LB medium with ampicillin, as falsly inoculated in Kanamycin

Agarosegelelectrophoresis with digestions

->Protocol (11 Agarose gel electrophoresis)

- Agarosegelelectrophoresis with the digestions (CMV, eGFP, pDS7, 190-6), 125V for 30 minutes and then for 20 minutes;

- expected DNA bands: 190-6 (4840bp, 1903bp), pDS7 (8027bp, 6bp), CMV (654 bp (Insert), 2079bp (Plasmid)), eGFP (720bp (Insert), 2750bp (Plasmid))

- Correct DNA bands for 190-6 (~4800bp, ~1900bp, ~6700bp (undigested plasmid)) and eGFP (~2000bp (Plasmid), ~750 bp (Insert)); CMV probably not digested (two bands; one probably normal, one supercoiled) and pDS7 not clear

Restriction digest from CMV and pDS7

-> Protocol (5 Restriction digest)

- Restriction digest from CMV (EcoR1, Pst1; 6µl DNA, buffer H) and pDS7 (EcoR1, Spe1; 2µl DNA, buffer B)

Agarosegelectrophoresis with digestions

->Protocol (11 Agarose gel electrophoresis)

- Agarosegelelectorphoresis for 30 minutes, 150V

- Expected DNA bands: CMV see above, pDS7 (3647bp, 3369bp, 1011bp, 6bp)

- false DNA bands CMV (~1200 bp, ~2000 bp) and pDS7 (~8000bp two bands, ~1100 bp); required to isolate a new colony for these two Plasmidextractions

Plated CMV on Ampicllin-Agar

- Plated the colony from CMV (BBa_J52034) for Plasmidextraction (Ampicillin), as falsly plated on Kanamycin

8-14-2010

weekend

8-15-2010

weekend

8-16-2010

Planting colonies

- transfer 1 ml PhiC31o culture to new LB medium + Amp, 37°C

- pick up CMV and pDS7 colonies from plates and transfer to LB medium+Amp, 37°C

Plasmid Extraction of PhiC31o

->Protocol (4 Plasmid extraktion from cells)

- plasmid extraction of PhiC310

->27,5ng/µl DNA and second plasmid extraction of PhiC310 (i. o. to get more DNA); first eluation-step with first eluation-extraction

-> 60ng/µl DNA

Restriction digest

->Protocol (5 Restriction digest)

- restriction digest of PhiC310 with EcoR1 and Spe1

H2Oddest, sterile 0 µl
Buffer B 2,0 µl
BSA (1:10) 2 µl
DNA (0,06µg/µl) 15,0 µl
EcoR1 0,5 µl
Spe1 0,5µl

restriction digest in the thermo cycler (program "Verdau", see protocol)

Handling primers after arrival (1,2,3,4,5,6,11,12)

->Protocol (9 Handling primers)

PCR preparations

- 10mM dNTP mix made from 100 mM dATP, dGTP, dCTP, dTTP by taking 100µl of each and adding 600µl H 2 O

PCR 1 and 6

- PCR of the tet inducible CMV minimal promotor out of prevTRE (=PCR 1 with Primer 1 and 2) and SV40PA out of pcDNA3 (=PCR 6 with Primer 11 and 12)

->Protocol (10 PCR with Pfu)

Mixture:

pTRERev (0,15µg/µl) pcDNA3 (0,6 µg/µl)
Primer 2*2,5µl (P1+P2) " (P11+P12)
300ng template 0,5µl 2µl
10x Buffer Pfu 5µl "
dNTP Mix 1µl "
Pfu Polymerase (3u/µl) 0,5µl "
H2O 40,5µl 39µl
summ 52,5µl 52,5µl


Programme:

Denaturation 95°C 2min
30 times: Denaturation 95°C 1min
Annealing 45°C 30sec
Extension 73°C 2min
Final Extension 73°C 5min
Soak (end) 12°C infinite

Glycerolstock of PhiC31o

- Glycerolstock of the colony of PhiC31o for the plasmidextraction

bacterial culture 800µl
Glycerol (50%) 500µl

8-17-2010

Agarose gel electrophoresis of PCR6 which shows that PCR6 is about 200bp

Plate CMV and pDS7 colonies on Ampicillin-Agar

- colonies for plasmidextraction of CMV and pDS7 plated on Ampicillinplates

Plasmid Extraction of CMV and pDS7

- plasmidextraction of CMV (2,5ng/µl) and pDS7 (10ng/µl) the A260/A280 value was 1.333, which means that it was 90% Protein and only 10% DNA (should be 1,8); new plasmidextraction needed

new overnight cultures of CMV and pDS7 for a new plasmidextraction made

Agarose gel electrophoresis

-> Protocol (11 Agarose gel electrophoresis)

- Agarose gel electrophoresis of the restriction digest of PhiC31o and PCR 1 and 6

- the right bands found for PhiC31o (~2900,~2400,~250)

- the right band found for PCR1 (~450)

- no band found for PCR6; new electrophoresis needed with more DNA loaded

Agarose gel electrophoresis of (from left to right) PhiC31o, PCR1 and PCR6 Agarose gel electrophoresis of (from left to right) PhiC31o, PCR1 and PCR6 which shows that PCR1 is between 250 and 500 bp

- new agarose gel electrophoresis from PCR6 with 5µl DNA instead of 3µl (image not yet shown)

- the right band found for PCR6 (~200)

New overnight cultures of CMV and pDS7

- the overnight colonies didn't grow; new colonies (CMV and pDS7) picked from plate and inoculated in LB Ampicillin

PCR purification of PCR 1 and 6

-> Protocol (12 Gel extraction or PCR Clean up)

- DNA concentration of the PCR 1 and 6 products measured: PCR1: 410ng/µl (A260/A280=1.253) PCR6: 568ng/µl (A260/A280=1.275)

- PCR Purification with Promega Kit

-> PCR1: 230ng/µl (A260/A280=1.769)

-> PCR6: 37.5ng/µl (A260/A280=1.667)

8-18-2010

Agarose gel electrophoresis of (from left to right) CMV and pDS7 showing the right bands for pDS7

Plasmid Extraction of CMV and pDS7

-> Protocol (4 Plasmid extraction from cells)

- Plasmid extraction of CMV (97.5ng/µl; A260/A280=1.857) and pDS7 (212ng/µl; A260/A280=1.848)

Restriction digestion

-> Protocol (5 Restriction digestion)

- Restriction digestion of CMV (EcoR1 + Pst1; 10µl DNA, buffer H) and pDS7 (EcoR1 + Spe1; 5µl DNA, buffer B)

-> expected DNA bands: CMV: 2079bp (plasmid) + 654bp (Insert); pDS7: 7022bp + 1011bp

Agarose Gel electrophoresis of digested CMV and pDS7

-> Protocol (11 Agarorse gel electrophoresis)

-> right DNA bands for pDS7 (~7000bp, ~1000bp)

-> false DNA bands for CMV

- Starting PCR 2a and 2b (replication and mutagenesis of pDS7): 3 µl DNA and 50°C Annealing Temperatur (other same as 8-16-2010)

8-19-2010

Agarose gel electrophoresis of PCR 2a and 2b

-> Protocol (11 Agarose gel electrophoresis) (150V, 30min)

Agarose gel electrophoresis of (from left to right) PCR2a and PCR2b
Agarose gel electrophoresis of (from left to right) PCR2a and PCR2b

-> the right bands for PCR2a (~300bp) and PCR2b (~700bp)

- New agarose gel electrophoresis with all of the PCR product for gel extraction (150V, 30min)

Gel extraction of the DNA from PCR2a and PCR2b

-> Protocol (12 Gel extraction or PCR Clean up)

- DNA concentration measured; problem with nanodrop as too low concentration; lyophille used to reduce volume

- DNA concentration measured again: PCR2a: 70ng/µl A260/A280=1.647; PCR2b: 45ng/µl A260/A280=1.5

PCR 3 (joining PCR of 2a and 2b)

- PCR3 (the joining PCR of PCR2a and 2b; Joining of the TEVrecogn-N-Degron-SF3 part) done: 1.3 µl of PCR2a and 4.7 µl of PCR2b makes 300ng of a 1:1 solution of both to be joined DNA parts. Annealing temperature: 50°C

-> Protocol (10 PCR with Pfu)

8-20-2010

Agarose gel electrophoresis of PCR3

Gelfoto from PCR3

left column: marker; rightmost column: PCR3

-> Protocol: 11 Agarose gel electrophoresis (150V, 30min)

-> expected band: ~1000bp

-> false band: ~500bp

- probable reason: mini photometre was influenced by gel extraction chemicals, therefore it measured false DNA concentrations and false template masses were calculated

-> New 2a and 2b PCR

New PCR (2a and 2b)

-> Protocol: 10 PCR with Pfu

(see 8-18-2010, but 35,5µl water)

8-21-2010

weekend

8-22-2010

weekend

8-23-2010

Agarose gel electrophoresis of PCR 2b

-> Protocol: 11 Agarose gel electrophoresis

- expected band: 700bp

-> no band shown on gel -> new PCR 2b

PCR 2b

- start PCR 2b with PCR 2b from 8-13-10 as template ( 1:20 and 1:100 diluted; 1µl)

-> Protocol: 10 PCR with Pfu

- annealing temperature: 50°C; amount of water: 37,5µl

Agarose gel electrophoresis of PCR 2b 1:20 and 1:100

-> Protocol: 11 Agarose gel electrophoresis

- expected bands: each ~ 700bp

- false bands: ~ 200bp

-> new PCR with 2ng, 5ng, 10ng template pDS7


PCR 2b with 2ng, 5ng, 10ng template pDS7

- pDS7 1:100 diluted(-> 2,1 ng/µl)

Mixture:

2ng 5ng 10ng
Primer 2*2,5µl (P5+P6) 2*2,5µl (P5+P6) 2*2,5µl (P5+P6)
10x Buffer Pfu 5µl 5µl 5µl
dNTP Mix 1µl 1µl 1µl
template pDS7 (dil.) 1µl 2,5µl 5µl
Pfu Polymerase (3u/µl) 0,5µl 0,5µl 0,5µl
DMSO 1,25µl 1,25µl 1,25µl
H2O 33,25µl 30,25µl 25,25µl
sum

-> Protocol: 10 PCR with Pfu

PCR 2a gel extraction

- Quiagen kit (QuiaexII)

-> Protocol: 14 QIAEX II gel extraction

Start 3 CMV overnight cultures

8-24-2010

agarose gel electrophoresis of PCR 2b

-> Protocol: 11 Agarose gel electrophoresis

Agarose gel electrophoresis of (from left to right) PCR2b (2ng (cut out), 10ng, 5ng template) showing the right bands for 2ng, 5ng template

- expected bands: right bands with 2ng and 5ng template (~700bp), no band with 10ng template

CMV plasmid extraction

-> Protocol: 4 Plasmid extraction from cells

Plasmid extractionof 3 different overnight cultures.

- results:

  • 52,5 ng/µl A260/A280= 1.312
  • 133 ng/µl A260/A280= 1.710
  • 80 ng/µl A260/A280= 1.600

CMV restriction digestion

-> Protocol: 5 Restriction digestion

- CMV restriction digest: EcoRI, PstI, buffer H

  • 19µl, H2O : 0µl
  • 6µl, H2O : 9.5µl
  • 12.5µl, H2O : 3µl

PCR 2b gel extraction

- PCR2b was gel extracted (with Qiagen gel extraction kit), 17.5 ng/µl a260/A280= 1.750

-> Protocol: 14 QIAEX II gel extraction

PCR 3 (fusion of 2a and 2b)

- PCR3: conducted again at 52°C annealing temperature

  • 10.5 ng (from PCR2b) 0.6µl
  • 4.5 ng (from PCR2a) 0.9µl (1:10 diluted)
PCR2a 0.9 µl
PCR2b 0.6 µl
primer3 2.5 µl
primer6 2.5 µl
dNTPs 1 µl
Pfu 0.5 µl
10xbuffer 5 µl
H2O 37 µl

-> Protocol: 10 PCR with Pfu

agarose gel electrophoresis of CMV digestion

- agarose gel electrophoresis (150V, 25 min) of the CMV digestion

-> bands are wrong again ( ~ 1200bp, 2000bp)

8-25-2010

Agarose gel electrophorese of PCR 3

-> Protocol: 11 Agarose gel electrophoresis

- expected band: ~1000bp

- false band: ~400bp

Plasmid extraction of ccdB tet and ccdB strep

Plasmid extraction of pSB1C3 with BBa_P1010

-> Protocol: 4 Plasmid extraction from cells

- results:

ccdB tet: 50ng/µl; A260/A280= 1,818


Plate ccdB amp, cam, tet

- plate ccdB with ampicilline, chloramphenicol, tetracycline resistance on LB agar with appropiate antibiotic.


Overnight culture of ccdB kan

- Overnight culture of ccdB with kanamycine resistance in LB medium with kanamycine

PCR 7a, 7b, 9, 10

->Protocol: 10 PCR with Pfu

PCR nr. template concentration dilution primer
7a 190-6 ~200ng/µl 1:100 13,14
7b 190-6 ~200ng/µl 1:100 15,16
9 eGFP 55ng/µl 1:25 20,21
10 PhiC31o 20ng/µl 1:10 22,23


Mixture

template (~2ng) 1µl
Pfu 0,5µl
Primer *2 2,5µl *2
10x buffer 5µl
dNTP Mix 1µl
H2O 37,5µl
sum 50µl

Standard PCR; annealing temperature: 60°C

8-26-2010

Agarose gelelectrophoresis of PCR 7a, 7b, 9, 10

->Protocol: 11 Agarose gel electrophoresis

- 150V, 25min

PCR nr. expected bands result
7a 850bp no band
7b 402bp false band (200bp)
9 808bp no band
10 1888bp no band

Plasmid extraction of ccdB kan

-> Protocol: 4 Plasmid extraction from cells

-result: concentration: 25ng/µl; A260/A280= 2,0

New PCR 7a, 7b, 9, 10

Mixture

template (~2ng) 1µl
Pfu 0,5µl
Primer *2 2,5µl *2
10x buffer 5µl
dNTP Mix 1µl
DMSO 1,25µl
H2O 36,25µl
sum 50µl

Program:

gradient PCR, 42-69°C annealing temp.

->Protocol: 10 PCR with Pfu

Overnight culture of ccdB amp, tet, cam

Inoculate one colony each in 5ml medium with approptraite antibiotic.

8-27-2010

Agarose gel electrophoresis of PCR 7a, 7b, 9, 10

->Protocol: 11 Agarose gel electrophoresis

150V, 25min, 75mA

gel electrophoresis of new PCR 7a,7b, 9, 10

from left to right: 7a, 7b, 9, 10, Marker

PCR nr. expected bands result
7a 850bp no band
7b 402bp right band (~400bp)+ false band (~150bp)
9 808bp false band (~200bp)
10 1888bp right band (~1900bp)+false band (~500bp)

Plasmid extraktion of ccdB amp, tet, cam

->Protocol: 4 Plasmid extraction from cells

results:

Plasmid concentration A260/A280
ccdB amp 57,5 ng/µl 1,917
ccdB cam 70,0 ng/µl 1,867
ccdB tet 50,0 ng/µl 1,818


New PCR 7a, 9

Mixture:

- 2ng template: see 26-8-10

- 4ng template: see 26-8-10, but 2µl template and 35,25µl water

-> Protocol: 10 PCR with Pfu


Restriction digestion of ccdB amp, kan, cam, tet

-> Protocol: 5 Restriction digestion

- only 90min 37°C incubation

- EcoRI, PstI, Buffer H

template volume mass
ccdB amp 16µl 930ng
ccdB cam 14,3µl 1µg
ccdB tet 16µl 800ng
ccdB kan 16µl 400ng

Agarose gelelectrophoresis of PCR 7a, 9, ccdB restriction digestion

150v, 25min, 75mA

-> Protocol: 11 Agarose gel electrophoresis

gel electrophoresis of new PCR 7a, 9, ccdb

results:

- PCR7a, 9: false band at 200bp

- ccdB: each digestion leads to a right band with ~ 650bp

8-28-2010

weekend

8-29-2010

weekend

8-30-2010

New PCR 7a and 9


Mixture

template (~4ng) 2µl
Pfu 0,5µl
Primer *2 2,5µl *2
10x buffer 5µl
dNTP Mix 1µl
DMSO 1,25µl
H2O 35,25µl
sum 50µl


-> Protocol: 10 PCR with Pfu


PCR program

PCR 7: Annealing Temperature 60°C - 25 x 1 min Annealing time and 5x 1,30 min Annealing time

PCR 9: Annealing Temperature 55°C - 25 x 1 min Annealing time and 5x 1,30 min Annealing time


Gel extraction of PCR 7b, 10

->Protocol: 14 QIAEX II gel extraction

results:

PCR nr. concentration A260/A280
7b 10 ng/µl 2,0
10 17,5 ng/µl 1,4


Agarose gel electrophoresis of new PCR 7a, 9

-> Protocol: 11 Agarose gel electrophoresis

150V, 25min

- results:

- 7a: no band shown

- 9: false band (~200bp)

New PCR 3

-> Protocol: 10 PCR with Pfu

PCR2a 0.9 µl
PCR2b 0.6 µl
dNTPs 1 µl
Pfu 0.5 µl
10xbuffer 5 µl
DMSO 1,25µl
H2O 36,75 µl
sum 45µl

- new method: standard PCR without primers (10 cycles, 56°C annealing temp.)

- then add 2,5µl of primer 3 and 6

- 30 cycles standard PCR (54°C annealing temp.)

8-31-2010

Gel photo of (left to right) PCR4a(2.5ng template), PCR4a(5ng template),PCR4b(2.5ng template), PCR4b(5ng template), PCR3(Pfu), PCR3(Phusion)

Agarose gel electrophoresis of PCR 4a, PCR4b, PCR3(Pfu), PCR3(Phusion)

-> Protocol: 11 Agarose gel electrophoresis

150V, 25min

- results:

- PCR4a(2.5ng template), PCR4a(5ng template),PCR4b(2.5ng template), PCR4b(5ng template), PCR3(Pfu): no band shown

- PCR3 (Phusion): right band (~1000bp)

New PCR PCR4a, PCR4b, PCR7a, PCR9

-> Protocol: 10 PCR with Pfu

PCR mixture for PCR4a, PCR4b

template 37.25 µl (200ng)
dNTPs 1 µl
Pfu 0.5 µl
10xbuffer 5 µl
DMSO 1,25µl
sum 50µl

Standard PCR program with annealing temperature PCR4a: 51.1°C, PCR4b: 48.5°C.

-> Protocol: 15 PCR with Phusion

PCR mixture for PCR7a, PCR9

template 4 µl (8ng)
dNTPs 1 µl
Phusion 0.5 µl
5xbuffer 10 µl
DMSO 1,25µl
H2O 28.25 µl
sum 50µl

PCR program: Phu62

98°C 1 min
98°C 10 sec
62°C 20 sec
73°C 30 sec
return to step 2 for 29 cycles
73°C 10 min
12°C forever


Gel photo of (left to right) PCR3, ladder, PCR7a, empty, PCR9

Agarose gel electrophoresis of PCR 3, PCR7a, PCR9 for gel extraction

-> Protocol: 11 Agarose gel electrophoresis

120V, 30min

- results:

- PCR3; right band (~1000bp) and side-product

- PCR7a: no band

- PCR9: right band (~800bp) and side-product

Gel extraction of the DNA from PCR3 and PCR9

-> Protocol (12 Gel extraction or PCR Clean up)

results:

PCR 9: 22,5ng/µl; A260/A280=1,8

PCR 3: 22,5ng/µl; A260/A280=2,25

New PCR 4a, 4b, 7a with DreamTaq

-> Protocol: 16 PCR with DreamTaq

PCR mixture for PCR7a

template 5 µl (10ng)
dNTPs 5 µl
DreamTaq 0.33 µl
10xbuffer 5 µl
DMSO 1,25µl
H2O 28.5 µl
sum 50µl

Primers for PCR 7a: 13,14

Annealing temp: 60°C

PCR mixture for PCR4a,4b

template 36,5 µl (180ng HeLa cDNA)
dNTPs 5 µl
DreamTaq 0.33 µl
10xbuffer 5 µl
DMSO 1,25µl
sum 50µl

PCR 4a

Primers for PCR 4a: 7,8

Primers for PCR 4b: 9,10

Annealing temp: 50°C

PCR program:

95°C 1 min
95°C 30 sec
50/60°C 30 sec
72°C 1 min (1kb/min)
return to step 2 for 29 cycles
72°C 10 min
12°C forever

9-01-2010

Agarose gel electrophoresis of PCR4a, PCR4b, PCR7 (DreamTaq)

-> Protocol: 11 Agarose gel electrophoresis

150V, 25min

results: no product

New PCR 4a, 4b with DreamTaq, Pfu, with concentration gradient and touch-down PCR

-> Protocol: 16 PCR with DreamTaq; 10 PCR with Pfu

PCR mixture for DreamTaq

Concentration Low Middle High
template 5 µl (1:1000) 31.75µl (1:1000) 2µl (1:10)
dNTPs 5 µl
DreamTaq 0.33 µl
10xbuffer 5 µl
DMSO 1,25µl
H2O 26.75 µl 0 29.75
sum 50µl

PCR mixture for Pfu

Concentration Low Middle High
template 5 µl (1:1000) 37.25µl (1:1000) 2µl (1:10)
dNTPs 1 µl
Pfu 0.5 µl
10xbuffer 5 µl
DMSO 1,25µl
H2O 32.25 µl 0 35.25 µl
sum 50µl

Primers for PCR 4a: 7,8; PCR 4b: 9,10

-> Protocol: Thermal cycler program: Touch down

Agarose gel electrophoresis of PCR4a, PCR4b

-> Protocol: 11 Agarose gel electrophoresis

150V, 25min

gel 9-1-10

from left to right: 4a: P1, P2, P3, D1, D2, D3; 4b: P1, P2, P3, D1, D2, D3

key:

"P"= PCR with Pfu

"D"= PCR with DreamTaq

"1"= low template concentration

"2"= middle template concentration

"3"= high template concentration

expected bands:

  • 4a: 330bp -> P2 and P3 show right bands and "primer clouds"(?)
  • 4b: 376bp -> P1, P2, P3 show right bands and "primer clouds" (?)

9-02-2010

Agarose gel electrophorese of PCR 4a P2, 4b P2

-> Protocol: 11 Agarose gel electrophoresis

- 120V, 45min, 1,5% Agarose gel

Agarose gel electrophoresis of (from left to right) PCR4aP2, Marker and PCR4bP2

Agarose gel electrophoresis of (from left to right) PCR4aP2, Marker and PCR4bP2


- Cut out bands at ~~ 350bp and extract

PCR Agarose gel extraction

-> Protocol: 14 QIAEX II gel extraction

results:

PCR nr. concentration A260/A280
4aP2 12,5 ng/µl 1,67
4bP2 72,5 ng/µl 1,53


New PCR 7a with Pfu and Phusion

template: 190-6, Primer 13,14

Mixture with Pfu

template (~2ng) 1µl
Pfu 0,5µl
Primer *2 2,5µl *2
10x buffer 5µl
dNTP Mix 1µl
DMSO 1,25µl
H2O 36,25µl
sum 50µl


-> Protocol: 10 PCR with Pfu

PCR mixture with Phusion

template (~2ng) 1,0 µl
dNTPs 1 µl
Phusion 0.5 µl
5xbuffer 10 µl
DMSO 1,25µl
H2O 31,25µl
sum 50µl

-> Protocol: 15 PCR with Phusion

New PCR 4a, 4b with Pfu

-> Protocol: 10 PCR with Pfu

-> Protocol: Thermal cycler program: Touch down

Mixture see 9-1-10, twice 4a and 4b

Agarose gel electrophorese of PCR 4a, 4b, 7a, gel extracted 4a and 4b

-> Protocol: 11 Agarose gel electrophoresis

- 25min, 150V

gel 9-2-10-2

from left to right: 4a*, 4a, 4b*, 4b, 7a Phusion, 7a Pfu, Ladder

-> result: 4b, 4b*: right bands (~330bp)

-remain: false bands/no band


gel 9-2-10-3

from left to right: ladder, 4 columns pathway, 4a gelextr., 4b gelextr.

-> results: slight right bands for 4a and 4b, no "primer clouds" anymore.

9-03-2010

Overlapping PCR 5 with Pfu and Phusion

template: 4a, 4b Primer 7,10

Mixture with Pfu

PCR4a (~15ng) 1.2µl
PCR4b (~15ng) 2µl (1:10)
Pfu 0.5µl
Primer *2 2.5µl *2
10x buffer 5µl
dNTP Mix 1µl
DMSO 1,25µl
H2O 34.05µl
sum 50µl

-> Protocol: 10 PCR with Pfu

PCR program: standard PCR program for Pfu, Annealing temperature: 54°C

PCR mixture with Phusion

PCR4a (~15ng) 1.2µl
PCR4b (~15ng) 2µl (1:10)
dNTPs 1 µl
Phusion 0.5 µl
5xbuffer 10 µl
DMSO 1,25µl
H2O 29.05µl
sum 50µl

-> Protocol: 15 PCR with Phusion

PCR program: standard PCR program for Phusion, Annealing temperature: 58°C

New PCR7a with Pfu

template: 190-6; Primer: 13,14

Mixture with Pfu

template (~2ng) 1µl
Pfu 0.5µl
Primer *2 2.5µl *2
10x buffer 5µl
dNTP Mix 1µl
DMSO 1,25µl
H2O 36.25µl
sum 50µl

-> Protocol: 10 PCR with Pfu

PCR program: standard PCR program for Pfu, Gradient: 54.4°C, 57.8°C, 61.4°C, 65.0°C

-> results:

-7a: only "primer cluods"

-5 Pfu: no defined product (slurred?)

-5 Phusion: "primer clouds"

9-04-2010

weekend

9-05-2010

weekend

9-06-2010

charges for sequencing

name 4a-7 4a-8 4b-9 4b-10 3-3 3-6 6-11 6-12
DNA 4a; 2.4µl 4a; 2.4µl 4b; 0.5µl 4b; 0.5µl 3; 2µl 3; 2µl 6; 0.5µl 6; 0.5µl
primer [1pmol/µl] 7; 3.2µl 8; 3.2µl 9; 3.2µl 10; 3.2µl 3; 3.2µl 6; 3.2µl 11; 3.2µl 12; 3.2µl
Tris (10mM); pH 8.2 1.4µl 1.4µl 3.3µl 3.3µl 1.8µl 1.8µl 3.3µl 3.3µl

every charge 7µl


New overlapping PCR 5 with Pfu

template: 4a, 4b; 15ng

Primer 7,10


Mixture

PCR4a (~7ng) 0.56µl
PCR4b (~8ng) 1.1µl (1:10)
Pfu 0.5µl
Primer *2 2.5µl *2
10x buffer 5µl
dNTP Mix 1µl
DMSO 1,25µl
H2O 36.6µl
sum 50µl

-> Protocol: 10 PCR with Pfu

PCR program: standard PCR program for Pfu, Annealing temperature: 46.1°C,48.5°C, 51.1°C

-> results: "primer clouds"


New PCR 7a with Pfu new dilution of 190-6; 1:100

template[2ng/µl] 1µl 2µl
pfu polymerase 0.5µl 0.5µl
primer13 2.5µl 2.5µl
primer14 2.5µl 2.5µl
buffer 5µl 5µl
dNTP Mix 1µl 1µl
DMSO 1,25µl 1.25µl
H2O 36.25µl 35.25
sum 50µl 50µl


Agarose gel electrophoresis of 7a

-> results: "primer clouds"


charges for sequencing for PCR 1,7b and 9

name 1-1 1-2 7b-15 7b-16 9-20 9-21 10-22 10-23
DNA 1[1:10]; 1.74µl 1[1:10]; 1.74µl 7b; 3µl 7b; 3µl 9; 1.78µl 9; 1.78µl 10; 3.8µl 10; 3.8µl
primer [1pmol/µl] 1; 3.2µl 2; 3.2µl 15; 3.2µl 16; 3.2µl 20; 3.2µl 21; 3.2µl 22; 3.2µl 23; 3.2µl
Tris (10mM); pH 8.2 2.06µl 2.06µl 0.8µl 0.8µl 2.02µl 2.02µl 0µl 0µl

every charge 7µl

9-07-2010

Agarose gel electrophorese of PCR gel extractions

150V, 25min

-> Protocol: 11 Agarose gel electrophoresis

-result:

gel photo

from left to right: PCR 1,2a,2b,3,4a,/,7b,9,10,/,ladder,pathway

PCR nr. 1 2a 2b 3 4a 7b 9 10
expected band (bp) 492 332 772 1087 330 402 808 1888
shown band(s) 550,200 300 750 1100 300 450 900,1500 1900
clean charge x x ~x x x x

new PCR for PCR6

PCR mixture for PCR6

Concentration Low High
template 1 µl (1:100) 15µl (1:100)
primer (11,12) 2.5 µl*2
dNTPs 1 µl
Pfu 0.5 µl
10xbuffer 5 µl
DMSO 1,25µl
H2O 36.25 µl 22.25 µl
sum 50µl

-> Protocol: Thermal cycler program: Touch down, 62°C-52°C, 30 cycles by 55°C

9-08-2010

Restriction digestion of eGFP, PCR6, ccdBamp

Gel photo of (left to right) PCR6(5ng/100ng) template

as prepatation for ligation

-> Protocol 5 Restriction digestion

eGFP SV40PA=PCR6 ccdBamp
DNA 5µl=300ng DNA 1µl=37,5ng DNA 2µl=115ng
Buffer MC 2µl Buffer D 2µl Buffer H 2µl
BSA 1:10 2µl BSA 1:10 2µl BSA 1:10 2µl
EcoRI 0,5µl XbaI 0,5µl EcoRI 0,5µl
SpeI 0,5µl PstI 0,5µl PstI 0,5µl
H2O 13µl H2O 14µl H2O 13µl
sum 23µl sum 20µl sum 20µl

1:30h 37°C, 20min 80°C

Agarose gel electrophoresis of PCR6

-> Protocol: 11 Agarose gel electrophoresis

25min, 150V


Ligation 1

eGFP 14,4µL (144ng)
SV40PA 4,8µL (48ng)
ccdBamp 2,9µL (100ng)
T4 Buffer 10x 3µL
T4 Ligase 0,5µL
H2O 4,4µL
sum 30µL


Ligation at 22,5°C for 30 min, denaturation at 65°C for 10 min.

Concentration of DNA in Ligation 1:

540ng A260/A280=2,4


Transformation

-> Protocol 18 competent cells2

The incubation time for the cells is here 1 hour.

9-09-2010

Agarose gel electrophoresis of PCR 1, 3, 4a, 5, 7a, 9 Agarose gel electrophoresis of PCR 1, 3, 4a, 5, 7a, 9

-> protocol 11 Agarose gel electrophoresis

results: "primer-clouds", PCR 9: no band


Plasmid extraction of ccdBcam, ccdBamp, Bak, CMV, PhiC31o

-> Protocol 4 Plasmid extraction from cells

Plasmid concentration [ng/µL] A260/A280
ccdBcam 22.5 1.0
ccdBamp 42.5 1.2
Bak 17.5 0.8
CMV 10.0 0.7
PhiC310 60 1.4

Eluated with H2O instead of the Eluation Buffer.


New PCR 1, 3, 4a, 5, 7a, 9, 10 with phusion

PCRnr. 1 3 4a 4b 5 7a 9 10
template pDS7 (1:100); 4µl 2a; 0.9µl+2b; 0.6µl Bak; 0.5µl Bak; 0.5µl 4a; 0.8µl+4b; 1µl 190-6 (1:100); 1µl eGFP (1:25); 2µl PhiC31o (1:10); 1µl
primer [10pmol/µl] 1,2; 2.5µl 3,6; 2.5µl 7,8; 2.5µl 9,10; 2.5µl 7,10; 2.5µl 13,14; 2.5µl 20,21; 2.5µl 22,23; 2.5µl
H2O 28µl 30.5µl 31.5µl 31.5µl 30.2µl 31µl 30µl 31µl
Annealing temperature 51.5°C 53.1°C 53.1°C 51.5°C 53.1°C 57.5°C 61°C 57.5°C

+ in each assay:

dNTP mix: 1µl, 5x Phusion buffer: 10µl, DMSO:1.5µl, Phusion:0.5µl

sum: 50µl

Program: standard Phusion PCR, 29 cycles; annealing temperatures: see above

-> Protocol: 15 PCR with Phusion

gel electrophoresis of PCR 1, 3, 4a, 5, 7a, 9, 10

[from left to right: PCR 1, 3, 4a, 5, 7a, 9, 10, Ladder]

PCR3, 9, 10 with right bands.

New PCR 1, 4a, 4b, 5, 7a with phusion

PCRnr. 1 4a 4b 5 7a
template pDS7 (1:10); 1µl Bak 1 ; 1µl Bak 1; 1µl 4a, 4b; 2 x 1.5µl pCT 190-6 (1:40); 1µl
primer 1, 2; 2 x 2.5µl 7, 8; 2 x 2.5µl 9, 10; 2 x 2.5µl 7, 10; 2 x 2.5µl 13, 14; 2x 2.5µl
H2O 31µl 31µl 31µl 30µl 30µl
Annealing temperature 52°C 52°C 48°C 50°C 55°C

+ each assay with dNTP-mix (1µl), 5xBuffer (10µl, DMSO (1.5µl), Phusion (0.5µl) -> sum: 50µl

-> Protocol Touch down 59 with phusion, 30 cycles with gradient appropriate for the annealing temperatures above.

9-10-2010

Agarose gel electrophoresis of PCR 1, 4a, 4b, 5, 7a

-> Protocol: 11 Agarose gel electrophoresis

gel electrophoresis of PCR 1, 4a, 4b, 5, 7a

[From left to right: Ladder, 1, 4a, 4b, 5, 7a]

Only 4a has been amplified successfully.


Agarose gel extraction of PCR 3, 4a, 5, 9, 10

gel electrophoresis of PCR 3, 4a, 5, 9, 10

from left to right: PCR 3, band ~700bp, PCR 4a, band ~300bp, PCR 5, bands ~550bp (5*), ~650bp (5), PCR 9, band ~800bp, PCR 10, band ~1900bp

results:

PCR 3 PCR 4a PCR 5 PCR 5* PCR 9 PCR 10
concentration [ng/µl] 20 10 5 30 35 25
A260/A280 1.6 1.333 2.0 2.0 1.750 2.0


-> Protocol 12 Gel extraction or PCR Clean up (Promega kit)

9-11-2010

weekend

9-12-2010

weekend

9-13-2010

charges for sequencing (retry of 6.9.)

name 4a-7 4a-8 4b-9 4b-10 3-3 3-6 6-11 6-12
DNA 4a; 2.4µl 4a; 2.4µl 4b; 0.5µl 4b; 0.5µl 3; 2µl 3; 2µl 6; 0.5µl 6; 0.5µl
primer [1pmol/µl] 7; 3.2µl 8; 3.2µl 9; 3.2µl 10; 3.2µl 3; 3.2µl 6; 3.2µl 11; 3.2µl 12; 3.2µl
Tris (10mM); pH 8.2 1.4µl 1.4µl 3.3µl 3.3µl 1.8µl 1.8µl 3.3µl 3.3µl

every charge 7µl


New PCR7a with Taq

template: p190-6

Primer 13,14

Mixture

template (~4ng) 2µl (1:100)
MasterMix for Taq 10µl
Primer *2 1.5µl *2
DMSO 0,5µl
H2O 4.5µl
sum 20µl

PCR program: touchdown PCR with Taq

1: 94°C 2'
2: 94°C 30"
3: 64°C/62°C/60°C/58°C/56°C/54° 30"
4: 72°C 2'
5: (for each temperature)repeat 2-4 2x
6: 94°C 30"
7: 58°C 30"
8: 72°C 2'
9: repeat 6-8 29x
10: 72°C 10'
11: 15°C break

-> Protocol: 21 PCR with Taq Mastermix

overnight culture inoculated of

CMV (amp)
ccdB (amp)
ccdB (cam)
pC31o (amp)
Bak (amp)


New PCR7a with Phusion Hot Start

with 2ng and 4 ng template

template 2ng 4ng
H2O 54µl 53µl
Buffer 5x 20µl 20µl
Primer (13,14) 2*10µl 2*10µl
DMSO 3µl 3µl
Hot Start 1µl 1µl

total: 100µl each, divided into 5 charges

program:

98°C 30sec
----
98°C 10sec
gradient: 50°C, 53°C, 56,6°C, 60,2°C, 64,5°C 30sec 30 cycles
72 15sec
----
72°C 5min
12°C forever

-> Protocol:20 PCR with Phusion Hot Start

9-14-2010

Agarose gelelectrophoresis of PCR 7a

-> Protocol: 11 Agarose gel electrophoresis

bands 1 to 5: 2ng of template DNA

bands 6 to 10: 4ng of template DNA

-> no bands

new PCR 7a and PCR 8 (without mutation) with mastermix, without DMSO

number: 7a diluted 7a undiluted 8
mastermix 50µl 50µl 10µl
primer 2*10µl 2*10µl 2*2µl
H2O 29µl 29µl 5µl
template 1µl 190-6 (1:100) 1µl 190-6 1µl 190-6 (1:100)

7a diluated and undiluated: divided into 3 charges

7a diluated: 1,2,3; 7a undiluated: 4,5,6

1 2 3 4 5 6 8
48°C 52°C 56,1°C 48°C 52°C 56,1°C 52°C

program:

94°C 2'
94°C 30"
48°C/52°C/56,1°C 30"
72°C 1,5'
(for each temperature)repeat 30x
72°C 5'

Plasmid extraction of ccdB (amp) and ccdB (cam)

-> Protocol: 4 Plasmid extraction from cells

results:

ccdB(amp): 105ng/µl A260/280: 2,00
cddB(cam): 102ng/µl A260/280: 1,952

9-15-2010

Agarose gelelectrophoresis of PCR 7a diluted &7a undiluted & "8"(without mutation)

-> Protocol: 11 Agarose gel electrophoresis

9 15 10 Gelphoto

from left to right: 7a: 1:100 diluted: 48°C, 52°C, 56.1°C, undiluted: 48°C, 52°C, 56.1°C, 8 without Mutation

weak right band (with fuzz)for 7a undiluated with best result for 56°C

-> new PCR 7a and "8"

with mastermix Taq

-> Protocol: 21 PCR with Taq Mastermix

template: 190-6, 1:10 and 1:5

temp: 56°C and 58°C


number 7a 7a "8" "8"
Mastermix 20µl 20µl 20µl 20µl
H2O 15µl 15µl 15µl 15µl
Primer (13,14) 2*2µl (13,14) 2*2µl (13,16) 2*2µl (13,16) 2*2µl
Template 190-6 (1:5) 1µl 190-6 (1:10) 1µl 190-6 (1:5) 1µl 190-6 (1:10) 1µl

2 charges each: one for 56°C and one for 58°C annealing temperature


program:

94°C 2'
94°C 30"
56°C/58°C 30"
72°C 1,5'
(for each temperature)repeat 30x
72°C 5'


Restriction Digestion of PCR 1 (17.8.), PCR 3 (10.9.), PCR 51 (10.9.), PCR 52 (10.9.), PCR 6 (17.8.), PCR 9 (10.9.), PCR 10 (30.8.) for ligation with vector

EcoR1 + Pst1 with Buffer H; 50ng DNA

1 3 51 52 6 9 10
H2O 13µl 12,5µl 5µl 13µl 13,5µl 13,5µl 12,5µl
Buffer H 2µl 2µl 2µl 2µl 2µl 2µl 2µl
BSA (1:10) 2µl 2µl 2µl 2µl 2µl 2µl 2µl
DNA 2µl 2,5µl 10µl 2µl 1,5µl 1,5µl 2,5µl
EcoR1 0,5µl 0,5µl 0,5µl 0,5µl 0,5µl 0,5µl 0,5µl
Pst1 0,5µl 0,5µl 0,5µl 0,5µl 0,5µl 0,5µl 0,5µl

-> Protocol: 5 Restriction digestion

Agarose gelelectrophoresis of PCR 7a 1-4 & "8" 1-4(without mutation)

-> Protocol: 11 Agarose gel electrophoresis

-> bad results

purification of restriction digestion

concentration (ng/µl) A260/A280 (supposed) lenght
1 20 1,000 492
3 5 1,000 1087
51 22,5 1,8 688
52 7,5 1,5 688
6 10 1,333 237
9 20 1,6 808
10 17,5 1,75 1888

-> Protocol: 12 Gel extraction or PCR Clean up (Promega kit)

Ligation with pSB1C3 (2072bp)

for # backbone insert (µl) charge (µl) Buffer 10x (µl) H2O (µl)
100ng 1 4 7,12 20 2 6,38
50ng 3 2 31,48 40 4 2,02
100ng 51 4 8,85 20 2 4,65
100ng 52 4 26,56 40 4 4,94
100ng 6 4 6,86 20 2 13,36
100ng 9 4 11,7 20 2 1,8
100ng 10 4 31,24 40 4 4,26

plus 1 µl T4 ligase in each charge

-> Protocol: 22 Ligation


new PCR 7a & "8"

with mastermix Taq 56°C

7a-1 7a-2 "8"-1 "8"-2
mastermix (µl) 10 10 10 10
template 190-6 1µl 190-6 2µl 190-6(1:10) 1µl 190-6(1:10) 2µl
primer 13&14 2*1µl 13&14 2*1µl 13&16 2*1µl 13&16 2*1µl
H2O 7µl 6µl 7µl 6µl

sum: 20µl each

pcr-program:

94°C 2'
94°C 30"
56°C 30"
72°C 1,5'
(for each temperature)repeat 30x
72°C 5'
12°C forever

-> Protocol: 21 PCR with Taq Mastermix

9-16-2010

Agarose gelelectrophoresis of PCR 7a-1, PCR 7a-2, PCR 8-1, 8-2 and of all three PCR 6 we ever made (in order to control whether we have extracted the right fragment (237) or just the primerdimers)

-> Protocol: 11 Agarose gel electrophoresis

9 16 10 Gelphoto

From left to right: 62, 61, 6, Ladder, 7a-1, 7a-2, 8-2, 8-1

results:
6-1 and 6-2: ?
6: ok
7a-1,7a-2,"8"-1,"8"-2: primerdimer-problem -> we ordered new primers!

transformation of the ligations 1,3,51, 52, 6, 9, 10

-> Protocol:3 Transformation

->plated on plates with chloramphenicol

again PCR 1,3,5,6,9,10 in order to increase the profit (Ausbeute) for ligation

1 3 5 6 9 10
template prev TRE(1:30) 1µl 2a(1:5) 1µl + 2b 0,5µl 4a 0,5µl + 4b 0,5µl SV40PA (pcDNA3)(1:50) 1µl eGFP(1:5) 1µl PhiC310(1:6) 1µl
primer 1&2 2*2,5µl 3&6 2*2,5µl 7&10 2*2,5µl 11&12 2*2,5µl 20&21 2*2,5µl 22&23 2*2,5µl
Tm 54°C 50°C 49°C 56°C 58°C 58°C
mastermix (µl) 25 25 25 25 25 25
DMSO 1,25µl 1,25µl 1,25µl 1,25µl 1,25µl 1,25µl
H2O (µl) 17,75 17,25 17,75 17,75 17,75 17,75

pcr-program:

94°C 2'
94°C 30"
Tm (see above) 30"
72°C 1,5'
(for each temperature)repeat 30x
72°C 5'

-> Protocol: 21 PCR with Taq Mastermix

9-17-2010

Analysis of the transformation from yesterday

Colonies on plates when 100µl or pellet plated:

1 3 5 6 9 10
pellet no yes yes yes yes yes yes
100µl no yes yes yes yes yes yes

again: transformation of ligation 1

->Protocol: 3 Transformation

Agarose gelelectrophoresis of yesterday's PCR 1,3,5,6,9,10(without mutation)

-> Protocol: 11 Agarose gel electrophoresis

9 17 10 Gelphoto

From left to right: PCR1, PCR3, PCR5, Ladder, PCR6, PCR9, PCR10

-> right band for PCR 3,5,6,9,10; no band for PCR 1

9 17 10 Gelphoto2

From left to right: Ladder, PCR3, PCR5, PCR9, PCR10

Bands took: 32: Band short over 1000bp, 31: Band short under 1000bp (probably 32 right), 5 the highest band, 9 upper band, 10 upper band

new PCR 1,5,6

1 5 6
template pTRE Rev 0,5µl 4a 1,5µl + 4b 1,5µl pcDNA3 0,5µl
primer 1&2 2*2,5µl 7&10 2*2,5µl 11&12 2*2,5µl
buffer 10x 5µl 5µl 5µl
dNTPs 1µl 1µl 1µl
Pfu 0,5µl 0,5µl 0,5µl
H2O 26,75 µl 24,25 µl 26,75 µl
T<sub<m</sub> 51°C 50°C 55°C

-> Protocol: pcr-program: standard Pfu 10 PCR with Pfu


Restriction digestion of PCR products, ccdB Plasmids and Biobricks for the 3A Method


Name H2O Buffer (each 2µl) BSA (1:10) DNA Volume DNA Mass Enzyms (2*0.5µl)
ccdBa 25 µl H 2µl 20µl ~600ng E+P
ccdBa 25µl H 2µl 20µl ~700ng E+P
1 43µl MC 2µl 2µl ~500ng E+S
3 25µl B 2µl 20µl ~200ng X+P
Primer 18+19 25µl B 2µl 10µl+10µl ? X+P
51 25µl MC 2µl 20µl ~100ng E+S
52 25µl MC 2µl 20µl ~600ng E+S
6 35µl B 2µl 10µl ~400ng X+P
9 25µl MC 2µl 20µl ~700ng E+S
10 25µl MC 2µl 20µl ~400ng E+S
eGFP 35µl MC 2µl 10µl ~550ng E+S
CMV1 35µl H 2µl 10µl ~530ng E+P

-> Protocol:5 Restriction digestion

Colony PCR of Ligations 3, 51, 52, 6, 9, 10 and PCR of CMV1 (to test if right)

10 1-4, 9 1-4, 6 1-4, 51 1-4, 52 1-4, 3 1-4 Colonies picked and put in following Mix:

PCR Mastermix 10µl
Biobrick Primer F 1.5µl
Biobrick Primer R 1.5µl
H2O 7µl

CMV1:

PCR Mastermix 10µl
Biobrick Primer F 1.5µl
Biobrick Primer R 1.5µl
Template 2µl
H2O 5µl

-> Protocol: 21 PCR with Taq Mastermix

Agarose Gel electrophoresis of 3, 51, 52, 6, 9, 10 and CMV1 (to test if right)

-> Protocol: 11 Agarose gel electrophoresis

17 9 10 Gelphoto

Above: From left to right: 3.1, 3.2, 3.3, 3.4, 51.1, 51.2, 51.3, 51.4, Ladder, 52.1, 52.2, 52.3, 52.4

Below: From left to right: 6.1, 6.2, 6.3, 6.4, 9.1, 9.2, 9.3, 9.4, Ladder, 10.1, 10.2, 10.3, 10.4, CMV1

-> right bands for 6.3 and 6.4, CMV1 ~1200bp (we think that this is right, as Biobrick sequenzing information indicates that it isn't 654bp but about 1200bp)


PCR clean up of PCR Gel extraction 31, 32, 5, 9, 10, PCR Product 6 and digestion PCR1, Primer 18+19, R51, R52, PCR6, PCR9, PCR10, PCR3

-> Protocol: 12 Gel extraction or PCR Clean up (Promega kit)

9-18-2010

weekend

9-19-2010

weekend

9-20-2010

Dephosphorylation of linearized ccdB amp and ccdB cam

Add 1µl TSAP to digested vectors.

Incutation: 37°C 15min; Inhibition: 74°C 15min

Ligation and 3A-Assemblies

Jump-or-Die Ligations: 1a, 1b (51),1b (52), 2, 3

Cut'N'Survive Ligations: 1a, 2b,

Biobricks for both Systems: CMV, PCR1 (tet-on-promoter)

Mixtures:

- 3A Assemblies (everything except CMV, PCR1):

Inserts: each 8µl; ccdB vector: 1µl; T4 Ligase: 0.5µl; T4 Ligase Buffer: 2µl; H2O: 1µl

- Biobricks:

Insert: 10µl; ccdB vector: 1µl; T4 Ligase: 0.5µl; T4 Ligase Buffer: 2µl; H2O: 7µl

Incubation: 2:30h 16°C; Inhibition: 10 min 65°C

->Protocols: 22 Ligation, 13 3A Method for Biobrick assembly

Colony PCR of PCR product ligations with pSB1C3

Mixture:

PCR Mastermix:130µl ; PrimerF:19,5µl ; PrimerR:19,5µl ; H2O:91µl

-> Protocol: 21 PCR with Taq Mastermix

Agarose Gel Electrophoresis of Colony PCRs

-> Protocol: 11 Agarose gel electrophoresis

Colony PCRs

above from left to right: pathway/ladder/3.5/3.6/3.7/3.8/51.5/51.6/51.7/51.8/52.5/52.6/52.7/52.8

below from left to right: ladder/6.5/6.6/6.7/6.8/9.5/9.6/9.7/9.8/10.5/10.6/10.7/10.8

9-21-2010

Agarose Gel Electrophoresis of PCRs 1, 5, 6, and Colony PCR 3.8, 51.7, 52.6, 9.7, 10.5, 10.6

-> Bad results, probably too much cells in PCR mix

-> Protocol: 11 Agarose gel electrophoresis

Colony PCR of 3.8, 51.7, 52.6, 9.7, 10.5, 10.6 again and of the 3A Ligations of yesterday as well as the Ligation of PCR1 and CMV with pSB1C3

PCR Master Mix: 60µl

Primer F: 9µl

Primer R: 9µl

H2O: 42µl

->22charges à 5µl

-> Protocol:21 PCR with Taq Mastermix


PCR 7a with new Primers

template 190-6 (1:10) 1µl
Master Mix 25µl
Primer 13k (short) 3,75µl
Primer 14 3,75µl
H2O 16,5µl

sum: 50µl

same with Primer 13l (long)

->Protocol: 21 PCR with Taq Mastermix

Agarose Gel Electrophoresis of Colony PCRs and PCR 7a

different ladder:

gelfoto 21 9 10 apo2


key: JD=Jump-or-Die; CS=Cut'N'Survive; first number=Ligation number (see schedule); second number=colonie number (marked on the plate)

from left to right:7ak(short primer), 7al(long primer), JD.1a.1, JD.1a.2,JD.1b51.1, JD.1b52.2, JD.1b52.1, JD.1b52.2, JD.2.1, JD.2.2, JD.31.1, JD.31.2, CS.1a.1, CS.1a.2


verified products:7a (~800bp); JD.2.1; JD.2.2(each ~1000bp);JD.31.1(~2000bp); CS.1a.2(~1500bp)

no bands shown: CS.2b.1, CS.2b.2, PCR1.1, PCR1.CMV,3.8, 51.7, 52.6, 9.7, 10.5, 10.6

-> Protocol: 11 Agarose gel electrophoresis

9-22-2010

Inoculate Colonies

inoculated in 4ml LB-medium with appropriate antibiotic

Plated residual transformated E. colis (9-20-10) (where we had few colonies on plates)

Colony PCRs

for 22*5µl charges:

PCR Mastermix 55µl
Primer F 8,25µl
Primer R 8,25µl
H2O 38,5µl
sum 110µl

-> Protocol: 21 PCR with Taq Mastermix

Annealing temperature: 53°C


PCR Purification of 7a

charges 7ak and 7al had not been labeled, so they were renamed.

results:

7a1: 135ng/µl; A260/A280=1.7

7a2: 133ng/µl; A260/A280=1.7


PCR8 with Phusion Hot Start II

-> Protocol: 20 PCR with Phusion Hot Start

template 7a 1:100 3µl ~4ng (850bp)
template 7b 1:10 2µl ~2ng (402bp)
Buffer 5X 10µl
Phusion 0.5µl
H2O 27µl
Primer 13 2.5µl
Primer 16 2.55µl
DMSO) 1.5µl
dNTP mix 1µl

Program: annealing temperature: 63°C, elongation time: 40sec

Agarose gel electrophoresis of Colony PCRs and PCR8

new ladder: fermentas FastRuler (TM) Middle Range DNA Ladder http://www.fermentas.com/en/products/all/dna-electrophoresis/fastruler-dna-ladders/sm111-fastruler-middle

gelfoto

some results:

PCR 8 (lanes 23, 24): no band

Colony PCRs CS.2b.10, PCR1cam.3, PCR1cam.4, CMVcam.4, CMVcam.4, JD.1b51.10 (lanes 7,8,9,10,11,14): show right bands



Plasmid extraction of "over-day" cultures ligation-7(=BB4),-8(=BB4),-9(=BB5),-12(=BB9) and pSB1C3

->Protocol: 4 Plasmid extraction from cells

concentrations (ng/µl - A260/280):

BB4(7):57,5 - 1,643
BB4(8):15 - 1,5
BB5(9):173 - 1,816
BB7(12):55,0 - 1,571
pSB1C3: 12,5 - 1,250

new PCR 7b in order to get better results

Master Mix 25µl
H2O 16,5µl
Primer 15 3,75µl
Primer 16 3,75µl
template 190-6 (1:10) 1µl

overnight-cultures

inoculated of: CS2bK10=BB11; PCP1K3+4=BB1; CMV-C-K3+4; JD1b51K10=BB2; pSB1C3

9-23-2010

Restriction digestion of BB5 for the 3A Method

Name H2O Buffer D BSA (1:10) DNA Volume DNA Mass Enzyms (2*0.5µl)
BB5 11 µl 2µl 2µl 4µl ~500ng X+P

sum: 20µl

-> Protocol:5 Restriction digestion

Plasmid extraction of overnight cultures BB2, BB11, CMV in cam and pSB1C3

->Protocol: 4 Plasmid extraction from cells

concentrations (ng/µl - A260/280):

BB2:232 - 1,603
BB11:170 - 1,889
CMV in cam:272 - 1,703
pSB1C3: 290 - 1,758

The pSB1C3 colony was pink (produced a red pigment or something like that, so we make a new transformation in order to extract only pSB1C3 and not some other vectors.

Agarose gelelectrophoresis of PCR 7b and pathway restriction digestions

-> Protocol: 11 Agarose gel electrophoresis

Gelphoto

From left to right: PCR7b, pathway (3x),ladder (5000,2000,850,400,100bp)

result: PCR 7b shows right band with ~400bp

PCR clean up of PCR 7b

-> Protocol: 12 Gel extraction or PCR Clean up (Promega kit)

result: 90ng/µl; A260/A280= 1,895


Restriction digestion of BB2 and BB11


Name H2O Buffer D BSA (1:10) DNA Volume DNA Mass Enzyms (2*0.5µl)
BB2 10 µl 2µl 2µl 5µl ~1.2mg X+P

sum: 20µl


Name H2O Buffer D BSA (1:10) DNA Volume DNA Mass Enzyms (2*0.5µl)
BB11 7 µl 2µl 2µl 8µl ~1.3mg X+P

sum: 20µl

-> Protocol 5 Restriction digestion


Ligation of BB1 and BB6

-> Protocol: 22 Ligation

Name H2O T4 Ligase-Buffer PCR1 Primer 18+19 ccdBAmp T4 Ligase
BB1 9.5 µl 2µl 5µl 2µl 1µl 0.5µl
Name H2O T4 Ligase-Buffer CMV E+S BB5 ccdBCam T4 Ligase
BB6 6.5 µl 2µl 8µl 2µl 1µl 0.5µl

PCR 8 with 7b(new) plus 7a-2 with Phusion Hot Start II; Primer 16 plus 13k and 13l

Buffer 5x 10µl
H2O 27µl
dNTPs 1µl
Primer 16 2,5µl
Primer 13k short 2,5µl
DMSO 1,5µl
template 7a(1:100)(850bp) 3µl -> 4ng
template 7b(new)(1:100)(402bp) 2µl -> 2ng
Phusion Hot Start II 0,5µl
->same with Primer 13l long<-

Overnight culture of PCR1 in cam- Vector

new inoculation of PCR1 in cam, Colony 3 in 5ml LB+cam

From left to right: PCR8k,PCR8l,ladder (5000,2000,850,400,100bp)

Transformation of pSB1C3, BB1, BB6

New transformation of pSB1C3 from Spring distribution because the old colony was pink.

Transformation of BB1 and BB6.


BB1 plated on amp-Agar, BB6 and pSB1C3 plated on cam-Agar

-> Protocol: 19 Transformation (Sina Science Services)

Agarose gelelectrophoresis of PCR8 (long and short primer 13)

PCR8k=short primer 13, PCR8l=long primer 13

-> Protocol: 11 Agarose gel electrophoresis

result: PCR 8k and 8l shows right band with ~1200bp

PCR purification of PCR 8

-> Protocol: 12 Gel extraction or PCR Clean up (Promega kit)

result: ~70ng/µl; A260/A280=1.75

9-24-2010

New "over-day" culture of pSB1C3

Inoculate one culture from the ligation yesterday in 4ml LB+cam

Ligation of BB72

Mixture:

Inserts: each 5µl; ccdB vector: 1µl; T4 Ligase: 0.5µl; T4 Ligase Buffer: 2µl; H2O: 7µl

Incubation: 5h 18°C; Inhibition: 20 min 65°C

->Protocols: 22 Ligation, 13 3A Method for Biobrick assembly

Colony PCRs of BB1 and BB6

2 Colonys were picked from each plate -> 4 charges

Mixture for 4 charges with 5µl:

PCR Mastermix 2x 10µl
Primer F 1.5µl
Primer R 1.5µl
H2O 7µl
sum 20µl

+ Colony

from left to right: 11, 12, 61 and 62

-> Protocol: 21 PCR with Taq Mastermix

Annealing temperature: 53°C, standard protocol

Plasmid extraction of new PCR1 in cam vector

-> Protocol: 4 Plasmid extraction from cells

results:

concentration 82.5ng/µl
A260/A280 1.737


Agarose Gelelektrophoresis of 11, 12, 61 and 62


Plasmid extraction of pSB1C3 in cam vector

-> Protocol: 4 Plasmid extraction from cells

results:

concentration 22.5ng/µl
A260/A280 1.5

9-25-2010

weekend

9-26-2010

weekend

9-27-2010

Colony PCR of BB72, BB6 and BB1 plus a negativ charge as reference

PCR Mastermix 17.5µl
Primer forward 2.625µl
Primer revers 2.7µl
H2O 12.1µl
sum 35µl = 5µl per charge

-> Protocol 24 Colony PCR


Ligation of BB10

CMV© E+S 5µl
PCR 8l X+P 5µl
ccdBamp 1µl
T4 Ligase-Buffer 2µl
T4 Ligase 0.5µl
H2O 6.5µl
sum 20µl

-> Protocol 22 Ligation


Ligation of BB001(PCR1 in ©), BB017(PCR5 in ©), BB018(PCR6 in ©) and BB021(PCR9 in ©)

ccdB 1µl
BB001/017/018/021 5µl
T4 Ligase-Buffer 2µl
T4 Ligase 0.5µl
H2O 11.5µl
sum 20µl

-> Protocol 22 Ligation

Restriction digestion of PCR32, 8l, 10 and Primer 18+19 with E+P

template H2O BSA [1:10] Buffer H EcoRI PstI
PCR32 10µl 5µl 2µl 2µl 0.5µl 0.5µl
PCR8l 10µl 5µl 2µl 2µl 0.5µl 0.5µl
Primer 18+19 2x7.5µl -- 2µl 2µl 0.5µl 0.5µl
PCR10 10µl 5µl 2µl 2µl 0.5µl 0.5µl

-> Protocol 5 Restriction digestion

9-28-2010

Plating BB017, BB021, BB018, BB001, BB72, BB10

Amp BB72 BB10
Cam BB001 BB017 BB018 BB021

Colony PCR BB1, BB6, BB72 with DreamTaq (each 2 colonies)

-> Protocol: 16 PCR with DreamTaq

PCR mixture for PCR7a

template pick up colony from plate
primers*2 0.25µl*2
dNTPs 0.5µl
DreamTaq 0.05µl
10xbuffer 0.5µl
H2O 3.45µl
sum 5µl

Primers for colony PCR: BBseqF, BBseqR

Annealing temperature: 53°C

Ligation of BB2, BB019, BB020, BB016, BB022

BB2 BB019 BB020 BB016 BB022 Volume
PCR 52 8 32 10 each 5µl
PCR 6 each 5µl
Primer 18+19 each 5µl
ccdBamp 1µl
ccdBcam 1µl 1µl 1µl 1µl
T4 Ligase-Buffer 2µl 2µl 2µl 2µl 2µl
T4 Ligase 0.5µl 0.5µl 0.5µl 0.5µl 0.5µl
H2O 6.5µl 11.4µl 11.4µl 11.4µl 11.4µl
sum 20µl 20µl 20µl 20µl 20µl

9-29-2010

plates BB72, BB017, BB10, BB021, BB001, BB018: nothing grown -> thrown into trash

colony-PCR of BB2/8, BB019, BB016, BB020, BB022 with Taq

->protocol: PCR with Taq

H2O 6,7µl
Buffer 10x 1µl
MgCl2 1µl
dNTPs 0,2µl
Primer forw. seq f 0,5µl
Primer rev. seq r 0,5µl
Taq 0,1µl

template: pick colony

sum: 10µl; we made 100µl ->10 charges (2 colonies of each plate)

tube numbers:

1 2 3 4 5 6 7 8 9 10
BB2/BB8 - 1 BB2/BB8 - 2 BB016 - 1 BB016 - 2 BB019 - 1 BB019 - 2 BB020 - 1 BB020 - 2 BB022 - 1 BB022 - 2

PCR-program: taq53

agarose-gelelektrophoresis -> no bands visible

new plates made of BB1, BB4, BB6, BB7

Ligations BB72, BB017 (mit PCR51), BB10, BB021, BB001, BB018

PCR (5 µl each) PCR (5µl each) ccdB(amp) ccdB© Buffer T4 Ligase H2O
BB72O 1 32 1µl 2µl 0,5µl 6,5µl
BB017 51 1µl 2µl 0,5µl 11,5µl
BB10 CMV© PCR8 1µl 2µl 0,5µl 6,5µl
BB021 9 1µl 2µl 0,5µl 11,5µl
BB001 1 1µl 2µl 0,5µl 11,5µl
BB018 6 1µl 2µl 0,5µl 11,5µl

restriction degestions

of PCR6, PCR52, PCR32, PCR1, ccdB(amp), ccdB(cam)

template digestion with volume
PCR 6 Xba1 & Pst1 I. 10µl II. 3µl
PCR52 EcoR1 & Pst1 20µl
PCR32 Xba1 & Pst1 10µl
PCR1 EcoR1 & Spe1 2µl
ccdB(amp) EcoR1 & Pst1 20µl
cddB(cam) EcoR1 & Pst1 20µl

->protocol: 5 Restriction digestion


colony-PCR of BB4/c3, BB6/c7, BB72/c5, BB1/c5, (BB11 as control) with Taq

->protocol: PCR with Taq

Mastermix:

H2O 32µl
Buffer 10x 5µl
MgCl2 5µl
dNTPs 1µl
Primer forw. seq f 2,5µl
Primer rev. seq r 2,5µl
Taq 0,5µl
DMSO 1,5µl
sum 50µl

->10µl for each charge

template: pick colony; BB11: 2ng

tube numbers:

1 2 3 4 5
BB4-c3 BB6-c7 BB72-c5 BB1-c5 BB11

PCR program:TA = 53°C; elongation time=1min

9-30-2010

Colony PCR of BB2/BB8, BB6 K7&K8 and test PCR of BB11 with new dilutions and primers

Colony/BB11 2.5µl
H2O 32µl
dNTP 1µl
Primer F 2.5µl
Primer R 2.5µl
MgCl2 5µl
DMSO 1.5µl
Buffer 10x (with KCl) 5µl
Taq Polymerase 0.5µl
sum 56µl

-> Protocol 24 Colony PCR

PCR purification of the digestions of 6I, 6II, 52, 32, 1

concentration [ng/µl] A260/A280
6II 32.5 1.444
ccdBamp 0.0 ---
ccdBcam 5.0 1.000
6I 0.0 ---
52 20.0 1.143
32 27.5 1.57
1 25 1.667

-> Protocol 12 Gel extraction or PCR Clean up (Promega kit)

Transformation of BB018, BB72, BB001, BB017, BB10 and BB021

-> Protocol 19 Transformation SSS

Plasmidextraction of the following overnight cultures: BB020(K1), BB018(K5), BB6(K5), BB022(K1), BB016(K1), BB019(K1), BB2(K1) and BB018(K3)

concentration [ng/µl] A260/A280
BB020(K1) 163 1.711
BB018(K5) 92.5 1.85
BB6(K5) 120 1.778
BB022(K1) 105 1.75
BB016(K1) 85 1.889
BB019(K1) 105 1.909
BB2(K1) 190 1.81
BB018 110 1.833

-> Protocol 4 Plasmid extraction from cells

10-01-2010

new test PCR of BB5 and BB11 with 20µl and 50µl volume

Master Mix

H2O 67µl
dNTP 2µl
Primer F 5µl
Primer R 5µl
MgCl2 10µl
Buffer 10x (with KCl) 10µl
Taq Polymerase 1µl
sum 100µl

template: BB5/50µl: 5ng, 20µl: 1.5ng; BB11/20µl: 3ng

->protocol: PCR with Taq

01.10.10. Gel photo

from left to right: ladder, BB5/50µl, BB5/20µl, BB11/20µl. BB5/50µl and BB11/20µl show the right band.

10-02-2010

weekend

10-03-2010

weekend

10-04-2010

sequencing

cycle, clean and run

CMV (c) PCR 1 (c) BB4 7 BB4 8 BB018K5 BB018K3 BB6K5 BB2K1
template 1µl 2µl 3µl 3,8µl 2µl 2µl 2µl 1µl
primer (1:100) 3,2 µl 3,2 µl 3,2 µl 3,2 µl 3,2 µl 3,2 µl 3,2 µl 3,2 µl
10mM TrispH 8,5 2,8 µl 1,8 µl 0,8 µl 0 µl 1,8 µl 1,8 µl 1,8 µl 1,8 µl
name of tube 028 FIR 001 F/R 4-7 F/R 4-8 F/R 018-5 F/R 018-3 F/R 6-5 F/R 2-1 F/R


PCR

019 022 016 020
template (1:100 = 2ng) 2µl 2µl 2,5µl 1,5µl
Primer F 1µl 1µl 1µl 1µl
Primer R 1µl 1µl 1µl 1µl
dNTPs 0,4µl 0,4µl 0,4µl 0,4µl
Taq 0,2µl 0,2µl 0,2µl 0,2µl
Buffer 2µl 2µl 2µl 2µl
MgCl2 2µl 2µl 2µl 2µl
H2O 11,4µl 11,4µl 10,9µl 11,
colour of tube orange rose green blue

10-05-2010

Colony PCRs of BB6, BB4, BB021, BB022, CMV©, BB001, BB016, BB1, BB252, BB72, BB017, BB018, BB019, BB020

agarose gel electrophoresis

schedule of the agarose gel electrophoresis:

gel 1 BB6K9 BB6K10 BB6K11 BBK12 BB6K13 BB4K5 ladder BB4K6 BB4K7
BB021K14 BB021K13 BB021K12 BB021K11 BB022K18 BB022K19 ladder BB022K17 BB022K16
gel 2 BB022K12 BB022K11 CMV©K4 CMV©K5 BB001K5 BB001K6 BB001K7 BB001K8 ladder
BB016K17 BB1K16 BB1K15 BB1K8 BB1K7 BB252K1 BB252K2 BB252K3 ladder
gel 3 BB017K14 BB017K13 BB017K12 BB017K11 BB018K17 BB018K16 BB018K15 BB018K14 ladder
BB019K6 BB019K5 BB019K4 ladder BB019K3 BB020K7 BB020K8 BB020K6 BB020K5
gel 1 BB4K8 BB4K9 BB4K10 BB021K10 BB021K15
BB022K15 BB022K14 BB022K13 --- ---
gel 2 BB001K9 BB001K10 BB016K11 BB016K12 BB016K13 BB016K14 BB016K15 BB016K16 ---
--- BB252K4 BB252K5 BB72K12 BB72K11 BB72K10 BB72K9 BB017K16 BB017K15
gel 3 BB018K13 BB018K12 BB018K11 BB018K10 BB018K9
BB020K4 BB020K3 --- ---

Gelbilder und Auswertung einfügen

9 15 10 Gelphoto


overnight cultures of BB6K3, BB4K10, CMV©K4, BB001K5, BB001K9, BB016K11, BB1K8, BB252K3, BB72, BB017K14, BB018K10, BB019K6

10-06-2010

Plasmidextraction of the following overnight cultures: BB6(K13), BB4(K13), CMVc (K4), BB001(K5), BB001(K9), BB016(K11), BB1(K8), BB2(K3), BB7(K10), BB017(K14), BB018(K10) and BB019(K6)

concentration [ng/µl] A260/A280
BB6(K13) 265 1.8
BB4(K10) 40 2.0
CMVc(K4) 97.5 1.8
BB001(K5) 30 1.7
BB001(K9) 17.5 1.7
BB016(K11) 45 1.7
BB1(K8) 57.5 1.9
BB2(K3) 60 1.8
BB7(K10) 30 1.7
BB017(K14) 12.5 1.5
BB018(K10) 35 1.8
BB019(K6) 37.5 1.5

-> Protocol 4 Plasmid extraction from cells

10-07-2010

colony PCR of BB021, BB022 with Taq (53°C)


Primer fw 0.5 µl
Primer rev 0.5 µl
dNTPs 0.2 µl
Taq 0.1 µl
10xbuffer 1 µl
MgCl2 1 µl
H2O 6.7 µl

10-08-2010

plasmide extractions from 10-06-2010 were put in a lyophilizer and redissolved in less water (10-20 µl)

concentration [ng/µl] A260/A280
BB4(K10) 258 1,981
BB001(K5) 325 1.94
BB001(K9) 205 1.907
BB7(K10) 170 1.744
BB017(K14) 215 1.955
BB018(K10) 500 1.88
BB019(K6) 293 1.95

10-09-2010

Plasmid extraction BB018K5, BB018K6, BB021K17 and BB021K24

concentration of these samples to be measured on Monday

-> Protocol 4 Plasmid extraction from cells

10-10-2010

weekend

10-11-2010

Plasmid extraction BB018K5, BB018K6, BB021K24 and BB021K17 from 10-09-2010


concentration [ng/µl] A260/A280
BB018K5 55 1.571
BB018K6 92,5 1.762
BB021K24 27.5 1.833
BB021K17 37.5 1.508


Colony PCR BB01, BB018, BB6, BB252

stripe 1 BB001K5 K6 K7 K8 K9 K9 K10 BB252 K2
stripe 2 BB252 K3 K4 K5 BB6 K9 K10 K11 K12 K13
stripe 3 BB018 K9 K10 K11 K12 K13 K14 K15 K16
single sample BB018 K17


gel picture colony PCR

foto einfügen!

Sequencing 8,9 and 10 (cycle, clean and run)

BB001K9 for BB001K9 rev BB4K16 for BB4K16 rev BB001K5 for BB001K5 rev BB252K3 BB252K3
template 1µl 1µl 1µl 1µl 0,5µl 0,5µl 3,8µl 3,8µl
primer (1:100) 3,2 µl 3,2 µl 3,2 µl 3,2 µl 3,2 µl 3,2 µl 3,2 µl 3,2 µl
10mM TrispH 8,5 2,8 µl 2,8 µl 2,8 µl 2,8 µl 3,3 µl 3,3 µl 0 µl 0 µl
name of tube 31-F 31-R 44-F 44-R 24-F 24-R 32-F 32-R


BB72K10 for BB72K10 rev BB017K14 for BB017K14 rev BB018K10 for BB018K10 rev BB019K6 for BB019K6 rev
template 1,5µl 1,5µl 1µl 1µl 0,5µl 0,5µl 0,8µl 0,8µl
primer (1:100) 3,2 µl 3,2 µl 3,2 µl 3,2 µl 3,2 µl 3,2 µl 3,2 µl 3,2 µl
10mM TrispH 8,5 2,3 µl 2,3 µl 2,8 µl 2,8 µl 3,3 µl 3,3 µl 3 µl 3 µl
name of tube 14-F 14-R 21-F 21-R 13-F 13-R 34-F 34-R


CMV for CMV rev BB018K5 for BB018K5 rev BB018K6 for BB018K6 rev BB021K17 BB021K17
template 2µl 2µl 3,8µl 3,8µl 2µl 2µl 3,8µl 3,8µl
primer (1:100) 3,2 µl 3,2 µl 3,2 µl 3,2 µl 3,2 µl 3,2 µl 3,2 µl 3,2 µl
10mM TrispH 8,5 1,8 µl 1,8 µl 0 µl 0 µl 1,8 µl 1,8 µl 0 µl 0 µl
name of tube CMV-F CMV-R 185-F 185-R 186-F 186-R 2117-F 2117-R


ligation BB020 BB022, Bb6 and BB52

-> Protocol 19 Transformation SSS

BB020 BB022 BB6 BB52
insert (5µl) 18+19 PCR 10 CMV PCR5<sub<2</sub>
insert (5µl) - - BB5 PCR6
ccdB (1µl) c c c a
buffer 2µl 2µl 2µl 2µl
T4-ligase 0,5µl 0,5µl 0,5µl 0,5µl
H2O 11,5µl 11,5µl 6,5µl 6,5µl


PCR to test self-made taq


template eGFP 1µl
Primer 20,21 each 1µl
buffer (10x) 2µl
MgCl 2µl
dNTPs 0,2µl
taq 0,3µl to 0,7µl (0,1µl steps)
H2O fill to 20 µl

anealing temperature 58°C, elongation time 30