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PCR problems..


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#1 George250

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Posted 05 April 2009 - 07:27 AM

I have a problem with PCR..

When I am testing primers (e.g for gene A) there is expression of gene A in my samples that contained cDNA (Thank God for that!), although I have bands (including primer dimer formation) in my RT-ve and RNA-ve controls (during conventional PCR).. What that means? Genomic DNA contamination?

On the other hand, when I used the same first strand cDNA that I used above to test other primers (e. g gene B ) I have no bands in my RT and RNA -ve controls as well as there is no primer dimer formation.. There is only expression - strong bands of gene B only in my samples that contained cDNA..

Could you please give me a clue ? Do I need to redesign primers for gene A? What else can go wrong? Do you think that real time PCR can overcome this problem?

I dont think that my samples have been contaminated because if i had contamination then i should have had bands in my RT -ve controls in both cases (gene A and B ) and ot only in gene A.. Do you think it has to do with optimization and maybe I need to increase/decrease annealing T and/or primer/Mg concentration?

I am very comfused... Because this problem appears only with primers for gene A and not all the rest that I am working with..

Thank you very much! I look forward for your advice!!

#2 bob1

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Posted 05 April 2009 - 04:25 PM

I have a problem with PCR..

When I am testing primers (e.g for gene A) there is expression of gene A in my samples that contained cDNA (Thank God for that!), although I have bands (including primer dimer formation) in my RT-ve and RNA-ve controls (during conventional PCR).. What that means? Genomic DNA contamination?

What I think you have is contamination with PCR product from gene A, but not gene B, though genomic DNA is also a possibility if your primers are not exon spanning.

#3 chrisbelle

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Posted 05 April 2009 - 11:53 PM

There is a very small possibility that in certain conditions Taq polymerase can RT and amplify RNA to DNA. But I don't think that is the case in your problem. Most possibly like Bob said you have an exon-spanning primer set for Gene A, and there is DNA contam in you sample. Try using a Bioanalyzer to run your RNA sample, or do DNase digestion for your RNA before you proceed to cDNA.
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#4 George250

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Posted 06 April 2009 - 02:28 AM

There is a very small possibility that in certain conditions Taq polymerase can RT and amplify RNA to DNA. But I don't think that is the case in your problem. Most possibly like Bob said you have an exon-spanning primer set for Gene A, and there is DNA contam in you sample. Try using a Bioanalyzer to run your RNA sample, or do DNase digestion for your RNA before you proceed to cDNA.



Thank you very much for your answer, although I already did DNase digestion.. Do you think that with this problem I can proceed to real time PCR? Or i need to make new primers?




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