I am transforming A. rhizogenes with the plasmid. After transformation colony pcr works with a single sharp band at the expected size. But when I perform PCR for the same colonies on replica/patch plate (grown after 2 days on proper selection) or the overnight grown liquid culture (as a template) the PCR does not show any amplification. What might be the reason for this confusing observation?
possibly the first positive pcr was because some carryover of the transformation template was present but the transformation was ,in fact, not successful. Then when you take a colony and grow it on there is not enough reagent present to amplify and the unsuccessful transform cannot amplify.
Colony PCR is unreliable. As Paul Rutland said, you most likely saw a false positive due to excess template. The best advice is to pick colonies, restreak and grow them up in a liquid culture, and use that for a standard plasmid prep & PCR. Never use colonies directly from the transformation plate.
If you use a antibiotics for selection, you need to pick up a small fragment of the edge of the mycelium. If you picked up the middle of the mycelium, the mycelium is mixed with wild-type and mutant.
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