25-7-11
Plates of transformants with PBAD-cI-LVA-DT and PBAD-RBS-GFP-DT vector look good, since the self ligation plates don't have much colonies!
10μl plate is empty on self ligation plate control! 90μl plate has a few colonies...
Colony PCR as usual
Digestion PBAD, PhybB and vectors: cI-LVA and RBS-GFP
PBAD:
15μl PBAD
1μl SpeI
2μl Tango buffer
2μ MQ water
PhybB
3μl PhybB
1μl SpeI
2μl Tango buffer
14μl 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 LasR-LVA-DT):
μl vector
1μl EcoRI
1μl XbaI
3μl Fast digest buffer
1μl Fast AP
μl MQ water
Ligation:
PBAD-LasR-LVA-DT
6μl PBAD
8.5μl cI-LVA-DT vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
2.5μl MQ water
PhybB-LasR-LVA-DT
6μl PhybB
8.5μl cI-LVA-DT vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
2.5μl MQ water
Ligate for 30 to 45 minutes
Transformation as usual
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