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| Hello, 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? Thanks, Wendy |
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| cdna , libraries , nested , pcr |
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