Traceless mutagenesis (galK positive/counterselection)

1 post / 0 new
SkylaIde's picture
Traceless mutagenesis (galK positive/counterselection)

Now I am doing BAC recombeneering using methog galK-Kn positive/counterselection. I have the clone with intermediate construct (BAC with MCMV genome that contains insert galK/Kn cassete in the distinct part of MCMV genome). And now I have to perform the second step of this method and put insted of this cassete final insertion. Here I have problems. I do everything according to the protocol (preparing electrocompetent cells, electroporation, cell recovery and their plating in minimal plates) but I don't have positive clones. Even more on my water control plates I get more colonies than on the plates with cells that were electroporated with final construct. And on the plates with noninduced cells also observed colonies. Negative clones contain galK/Kn cassete, although they are not supposed to grow on the medium with DOG. I have repeated this procedure several times and every time I got approximately the same results - clones are negative but contain galK/Kn cassete.
If someone can give me advice and help in this situation I will very thanksfull. Hope for the best and thanks a lot for advance.