Plasmid pCP20 problem - (Jan/12/2009 )
I’m a student working in a research lab using the plasmid pCP20 which carries FLP recombinase and contains an ampicillin resistance cassette. We use this to remove knock out a resistance gene from S. Typhimurium. The Salmonella contains a kanamycin resistance gene. The protocol I have been using this far is as follows:
- Electroporation of competent Salmonella cells with pCP20 and recovery in SOB at 30°C for 1-2 hours in shaking incubator. Plate out onto agar and leave overnight (agar enriched with ampicillin present on plasmid to show successful transformation)
- Pick colonies and grow in antibiotic free broth at 43°C overnight (this is meant to cure plasmid from cells)
- Streak loopful of liquid culture and incubate overnight on LB agar at 37°C
- Screen for sensitivity of kanamycin and ampicillin antibiotic agar
I have a good prep of pCP20 to start with. I’ve managed to transform into Salmonella ok but then do not get successful removal of the kanamycin cassette.
Does anyone have any ideas/tips to make this work?
- Electroporation of competent Salmonella cells with pCP20 and recovery in SOB at 30°C for 1-2 hours in shaking incubator. Plate out onto agar and leave overnight (agar enriched with ampicillin present on plasmid to show successful transformation)
- Pick colonies and grow in antibiotic free broth at 43°C overnight (this is meant to cure plasmid from cells)
- Streak loopful of liquid culture and incubate overnight on LB agar at 37°C
- Screen for sensitivity of kanamycin and ampicillin antibiotic agar
I have a good prep of pCP20 to start with. I’ve managed to transform into Salmonella ok but then do not get successful removal of the kanamycin cassette.
Does anyone have any ideas/tips to make this work?
I am not familiar with this protocol, but I would like you to make sure that in the second overnight culture the cells are supposed to be grown at 43'C overnight and not 37'C overnight. For FLP recombinase activitiy in my E.Coli, I generally induce them with Arabinose (arabinose inducible promoter), so I am not sure. Although your FLP may be inducible by 43'C, are you sure it is overnight and not one hour or something, followed by 37'C overnight?