IGEM:Groningen/Notebook/iGEM 2011/2011/07/29: Difference between revisions

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==Entry title==
==29-7-11==
* Insert content here...
<br> Checking transformants of yesterday with colony PCR:
<br>Mastermix (9+1 samples):
<br> 10× Taq buffer: 40μl
<br> dNTP mix 10mM: 8μl
<br> MgCl2: 24μl
<br> taq 5u/μl: 1μl
<br> Forward primer BB vector 10μM: 8μl
<br> Reverse primer BB vector 10μM: 8μl
<br> MilliQ water: 310μl
<br> PCR conditions:
<br> 1. Pre heated lid: 111 °C
<br> 2. Denaturation: 10 min. at 94°C
<br> 3. Cycle 33×:
<br>      Denaturation: 30s at 94°C
<br>      Annealing:    30s at 60°C
<br>      Extension:    2.5 min. at 72°C
<br> 4. Final extension: 10 min. at 72°C
<br> 5. Store infinite at 4°C
<br>
<br> Use the overnight ligation mixture of yesterday for second transformation(just in case)...
<br> Procedure before storage overnight:
<br>PBAD 1:
<br> 16μl PBAD
<br> 1μl SpeI
<br> 2μl Tango buffer
<br> 1μl MQ water
<br>
<br>PBAD 2:
<br> 10μl PBAD
<br> 1μl SpeI
<br> 2μl Tango buffer
<br> 7μ MQ water
<br>
<br> After 2h: put 2.5μl Tango buffer+ 1μl EcoRI in these samples and digest for another 2h.
<br> DNA clean up with kit of Roche.
<br>
<br> Digestion vector (with RBS-GFP-DT):
<br>
<br> 3μl vector
<br> 1μl EcoRI
<br> 1μl XbaI
<br> 3μl Fast digest buffer
<br> 1μl Fast AP
<br> 21μl MQ water
<br>
<br> Ligation:
<br> PBAD(1)-RBS-GFP-DT
<br> 7.1μl PBAD
<br> 8.5μl RBS-GFP-DT vector
<br> 2μl T4 DNA ligase buffer
<br> 1μl T4 DNA ligase
<br> 1.4μl MQ water
<br>
<br> PBAD(2)-RBS-GFP-DT
<br> 7.8μl PhybB
<br> 8.5μl RBS-GFP-DT vector
<br> 2μl T4 DNA ligase buffer
<br> 1μl T4 DNA ligase
<br> 0.4μl MQ water
<br>
<br> Self ligation RBS-GFP-DT
<br> 8.5μl RBS-GFP-DT vector
<br> 2μl T4 DNA ligase buffer
<br> 1μl T4 DNA ligase
<br> 8.5μl MQ water
<br>
<br> Ligate for 30 to 45 minutes
<br>-> After this overnight ligation
<br>
<br> Transformation as usual
<br>
<br>
<br> Checking sequencing results AraC/PBAD-cI-LVA-DT
<br> AraC= OK, cI-LVA=OK. PBAD cannot be seen since the sequencing results go to a maximum of 1000 bp...
<br> Check in another way to detect the PBAD sequence, maybe primer design?
<br>
<br>




Revision as of 04:49, 29 July 2011

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29-7-11


Checking transformants of yesterday with colony PCR:
Mastermix (9+1 samples):
10× Taq buffer: 40μl
dNTP mix 10mM: 8μl
MgCl2: 24μl
taq 5u/μl: 1μl
Forward primer BB vector 10μM: 8μl
Reverse primer BB vector 10μM: 8μl
MilliQ water: 310μl
PCR conditions:
1. Pre heated lid: 111 °C
2. Denaturation: 10 min. at 94°C
3. Cycle 33×:
Denaturation: 30s at 94°C
Annealing: 30s at 60°C
Extension: 2.5 min. at 72°C
4. Final extension: 10 min. at 72°C
5. Store infinite at 4°C

Use the overnight ligation mixture of yesterday for second transformation(just in case)...
Procedure before storage overnight:
PBAD 1:
16μl PBAD
1μl SpeI
2μl Tango buffer
1μl MQ water

PBAD 2:
10μl PBAD
1μl SpeI
2μl Tango buffer
7μ MQ water

After 2h: put 2.5μl Tango buffer+ 1μl EcoRI in these samples and digest for another 2h.
DNA clean up with kit of Roche.

Digestion vector (with RBS-GFP-DT):

3μl vector
1μl EcoRI
1μl XbaI
3μl Fast digest buffer
1μl Fast AP
21μl MQ water

Ligation:
PBAD(1)-RBS-GFP-DT
7.1μl PBAD
8.5μl RBS-GFP-DT vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
1.4μl MQ water

PBAD(2)-RBS-GFP-DT
7.8μl PhybB
8.5μl RBS-GFP-DT vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
0.4μl MQ water

Self ligation RBS-GFP-DT
8.5μl RBS-GFP-DT vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
8.5μl MQ water

Ligate for 30 to 45 minutes
-> After this overnight ligation

Transformation as usual


Checking sequencing results AraC/PBAD-cI-LVA-DT
AraC= OK, cI-LVA=OK. PBAD cannot be seen since the sequencing results go to a maximum of 1000 bp...
Check in another way to detect the PBAD sequence, maybe primer design?


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