Joyce
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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