nested PCR from cDNA libraries

Wendy Grus wgrus at
Thu Mar 9 11:51:29 EST 2006

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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