*8th of July
Colony PCR of transformants to check if any transformant contains the plasmid with cI-LVA and LasR-LVA.
20μl total volume was used and the PCR was done with taq polymerase.
PCR program:
Denaturation: 95 °C for ten minutes
Cycle (33×):
Denaturation: 95°C for 30 seconds
Annealing: 60°C for 30 seconds
Extension: 72°C for 2 minutes
Final extension: 72°C for ten minutes
Store at 4 °C infinite
PCR with the new pBAD primers (dissolved and dilution made by me)
PCR was done in total colume of 50μl and with tag and Pfu polymerase (ratio 6:1) so Pfu can correct the mistakes that taq made
Denaturation: 94°C for ten minutes
Cycle (30×)
Denaturation: 94°C for 30 seconds
Annealing: 62°C and 65°C for 30 seconds
Extension: 72°C for 2 minutes
Final extension: 72°C for ten minutes
Store at 4 °C infinite
Check them on agarose gel 1% (TBE).
After that: Clean up the pBAD samples and nanodrop the samples
Nanodrop results:
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