I have been having problems with my PCR. I extracted genomic DNA and trying to make sure that I have the correct insert. I have done a control with actin to make sure that I have genomic DNA extracted, which was my positive control. I also used the primers to check for the insert and some of the candidates worked and some have not worked.
I wanted to have all four primers together to make sure that the negative results of some of my samples were not due to an error in the PCR.
Since the Tms were almost the same I used all 4 primers together, and used one sample that worked for the insert and one that didn’t. But this time I only see the actin band even for the candidate that worked for the insert my first trial.
Can some one advice me on how I should do this PCR including all 4 primers together.
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