Hello everyone!
I'm trying to knock out a non coding sRNA gene from E. coli genome by homologus recombination based method. I'm using Kan cassette and pSIM6 recombination system. I grow the pSIM6 strain at 30 degree till 0.5-0.6 OD and then transferred to 42 degree water bath with continuous shaking for 15mins. After giving induction, I transfer the cells in slurry ice with continuous swirling for 10 mins. Then the electro competent cells are made by giving two consecutive washing with nuclease free water. The generated DNA cassette is electroporated by using 0.2 cm cuvettes with 2.5kV voltage for 5.5ms. After the transformation, I grow the cells at 30 degree. I get 5-6 colonies. However, after doing colony PCR with the primers for the sRNA gene, I always get band in its proper size, which implies that the mutation did not occur. I have done several times, still not getting positive result. Please suggest how to troubleshoot.
Hello,
Did you add kanamycin to the medium for selection?
have you checked the competence of your bacterial host cells?
Did you do a homologous double or single recombination to get the knockout? Sometimes it's difficult to get the knockout, I had this experience, you had to do it several times.
Consider that the colonies on the plates may be mixtures of wt and mutant. So if even some cells are wt you will get the wt band after PCR. It might be better to look specifically for the mutant signal.
You might try to restreak your 5-6 colonies on kanamycin plates and test 1 or 2 individual colonies from each of those.
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