I'm using pCP20 vector carrying gene for FLP recombinase to remove kanamycin resistance cassette from bacteria genome. I know that there are two version of this vector: temperature or L-arabinose inductable (I use the secon one). After following all procedure I didn't obtain kanamycin resistance cassette removing from genome. If anyone use the same vector and could give me some clue I would be very gratetuf. Description of my procedure:

I transformed my bacteria with pCP20, incubate in 30C/180 rpm 2h, then spread them on MSB+Amp plates - they grown about 24h. Then I picked some colonies and put each of them (6 clones) to separated liquid culture (MSB+Amp) and incubated them ON/30C.180rpm. Next day I diluted culture 20x and incubated in 30C/180rpm with 0,2% L-arabinose in MSB+Amp thill they reach OD=1 (I added L-arabinose when OD was 0 and they grown about 6-7h), then I diluted them 20x and incubated them in 42C/220 rpm in MSB without antibiotics. After that, I diluted bacteria 1 mln times and spread them on MSB plate and incubated them ON. Next, I picked 80 colonies and patched them on: Km+MSB, Amp+MSB, MSB plates (in this order) and incubate Km+MSB and MSB at 37C and Amp+MSB at 30C ON. On this step I didn't obtain any colonies in Amp+MSB but all my clones grown in Km+MSB which means that they didn't loose kanamycin resistance cassette. I tried to change L-arabinose concentration to: 0,1 ; 0,5 or 3% but I obtain exactly the same result.

Edyta