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nested PCR from cDNA libraries

nested PCR from cDNA libraries - Protocols and Methods Forum

nested PCR from cDNA libraries - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.

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Old 03-09-2006, 04:51 PM
Wendy Grus
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Default 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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