July 05th, 2010
LuxAB - J61002(containing constitutive promoter J23106) transformation resulted in two useful colonies out of 4 colonies; LuxAB - P18 transformation resulted in 1 useful colony; I recultured the three useful colonies (one LuxAB - P18, two LuxAB - J61002).
Once recultures were grown enough I purified plasmid with High Pure Plasmid Purification Kit by Roche.
I extracted a total of 6 samples:
LuxAB - J61002 Colony 1 1
LuxAB - J61002 Colony 1 2
LuxAB - J61002 Colony 2 1
LuxAB - J61002 Colony 2 2
LuxAB - P18 1
LuxAB - P18 2
These are the order of the lanes of agarse gel 0.8% I ran to see if there were plasmids.
All 6 samples showed plasmids, now I'm gonna run a PCR to see if the insert of LuxAB is in the two vectors.
I ran a PCR with Taq Polymerase with 40 cycles:
5min 94°C --- | --- 45seg 94°C --- 45seg 55°C --- 3min 72°C (40 cycles) --- | --- 5min 72°C --- 4°C ->