Im working with b. Subtilis and trying to insert a chimeric gene into thymidelate A and thymidelate B. I already have the thyA mutant but when i transform my plasmid to mutate ThyB and plate them on SMM agar supplemented with thymine, casamimo acids and trimethroprim 10ug/ml and i do a colony pcr i see both the wild type and the mutant band. I restreaked these colonies and 24h later when i repeat the pcr i now see only the wild type band, so it seems like the mutants are dying. Any suggestions?
are you running a negative pcr control with no template dna and is the result of this sample negative as expected? I am concerned that you may be picking up pcr contamination from previous pcrs of the wild type and that you have the mutant present but the contamination has taken over the pcr reaction. Do the 2 pcrs use the same primers?
uhHi Paul. I always have negative and positive control. I don't see the same profile with them. I also use different PCR mix. I have this problem for about 4 month. That the mutant band loses intensity and eventully you only see wildtype. It seems something is lacking in the smm agar and thus the mutants are dying over the time. This mutation disrupt folate pathway.
that's good Sia . That does take the pcr out of the problem solving so hopefully someone can come up with a biochemical solution to this problem
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