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cDNA PCR help!

cDNA PCR help! - PCR - Polymerase Chain Reaction Forum

cDNA PCR help! - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 07-16-2009, 09:16 PM
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Default cDNA PCR help!



I'm trying to amplify a gene from cDNA collected from cells but it does not seem to be working. cDNA quality is fine since PCR using control primers (GAPDH) showed nice bands. However the primers I designed (used the NCBI primer designing tool with refseq as a template) never show any product. I've tried multiple annealing temperatures (from 51 to 58) and the best I've gotten is the smudge of primer dimers. There isn't even any streaking or smears. The product length is 600 bp so I don't know why this isn't working. I'm using hotstart Taq. Any help would be greatly appreciated!
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Old 07-17-2009, 08:41 AM
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Default Re: cDNA PCR help!

if you have determine there nothing wrong with the primer and all the parameter just don't seem to work. most likely is there some problem with ur primer stock. u may need to order it again. and was the reverse primer reserve complimented?

if possible can tell us what is ur MgCl2 conc and ur annealing and extension time?
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Old 02-04-2013, 09:05 AM
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Default Re: cDNA PCR help!

PCR (qPCR), has quickly become instrumental for quantifying DNA and for quantifying RNA in gene expression studies by reverse-transcription qPCR (RT-qPCR). But the quality of the results strongly depends on one of the preparatory steps: synthesis of cDNA. For example, in gene expression analysis, the starting RNA sample must be converted into cDNA before qPCR can begin. Yet many researchers do not realize the implications of this step in the process.
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