nested PCR from cDNA libraries
I used nested PCR with degenerate primers to determine a sequence of
interest from genomic DNA. With that sequence I designed specific
primers to amplify this sequence from cDNA libraries. However, the
specific primers will not amplify my sequence of interest. When I do a
nested PCR with first the outside degenerate pair and then the specific
pair, I do get product. Can anyone tell me what is going on? I am able
to amplify from the cDNA library with positive control actin primers, so
I know there is cDNA template. Is it just the quantity of the template
for my sequence of interest is really low?
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