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PCR gene specific amplification problem

PCR molecular biology markers

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#1 saripalligautam

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Posted 01 September 2013 - 09:59 PM

Hii everyone.

I am trying to amplify a gene specific fragment in wheat genome.Initially i used to get a very good amplicon as a PCR product but after some time , i started observing only a bright smear as a product.I tried all the possible modifications (like gradient, touchdown PCR, modifications in PCR mastermix, etc.) but in vain. Even i re synthesized my primers and also checked my DNA quality and quantity, and it's fine.I also used the same DNA with other primers and i am getting a good result.Can anyone extend the possible solutions to this problem?The below is the gel image of my previous and latest PCR amplification.

  

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#2 bob1

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Posted 03 September 2013 - 02:35 AM

It sounds like the DNA is degrading - which may not be visible on a gel and may not affect other primers depending on the abundance of the target.  It could also be that there is some inhibitor in there, but this would usually affect other reactions also.  Could you get a new lot of DNA and try that?



#3 saripalligautam

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Posted 17 September 2013 - 07:12 PM

It sounds like the DNA is degrading - which may not be visible on a gel and may not affect other primers depending on the abundance of the target.  It could also be that there is some inhibitor in there, but this would usually affect other reactions also.  Could you get a new lot of DNA and try that?

Thanks bob. We have tried even a new lot of DNA but still the same problem persists. Even tried with new set of primers but in vain.



#4 saripalligautam

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Posted 17 September 2013 - 07:12 PM

Thanks bob. We have tried even a new lot of DNA but still the same problem persists. Even tried with new set of primers but in vain.







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