IGEM:Groningen/Notebook/iGEM 2011/2011/06/14

14-6-11

Checking transformants/ testing whether DNA purification from gel went well and is functional in cloning:
PlasB from gel+ vector from gel: no colonies, maybe 1 or 2
PlasB from gel+ vector no gel: Lots of colonies, might be a lot of self ligation, but the self ligation control is negative...
PBADaraC from gel+ vector from gel: no colonies, just a few but it looks like contamination?
PBADaraC no gel+ vector gel: much colonies
PBADaraC gel + vector no gel: lots of colonies

Grow colonies , 3 colonies per different construct for plasmid prepping

Colony PCR
Taq 10× buffer: 40μl
dNTPs 10mM: 8μl
Forward Biobrick primer: 8μl
Reverse Biobrick primer: 8μl
Taq DNA polymerase: 2μl
MQ water: 334μl

PCR conditions:
Preheated lid: 111°C
Denaturation: 94°C for 10 min.
Cycle 33×:
denaturation: 94°C for 30s.
annealing: 60°C for 30s.
Extension: 72°C for 2,5 min.
Final extension: 72°C for 10 min.
Store infinite at 4°C

Analyse on a 1% TBE agarose gel.