PCR amplification in nontemplate control

2 posts / 0 new
Last post
sagarvarankar
sagarvarankar's picture
PCR amplification in nontemplate control

I have been facing a wierd problem where in I observe amplification in the non - template control. i know that this can be attributed o contamination in my reagents, however, I have checked all the possibiliies and am unable to find the problem. I'll briefly explain teh problem:
I am trying to determine gene copy number in the genomic DNA samples with the aid of qPCR [SyBr green chemistry]. First time when I set up this reaction along with a non - template control I observed amplification in the non - template control. Considering that the PCR was set with sterile milli Q water and not Sterile Nuclease free water [NFW] I used a fresh vial of Sterile NFW and obtained similar results. I also did a melt curve analysis to consider the possibility of primer dimer formation and did not obtain any such curve. Further I setup a standard PCR for the same reaction with completely different chemicals as I used a SyBr green mastermix for qPCR. Upon amplifying the product I observed a band in the non - template control at the exact location as that of my sample. To determine if any of my PCR chemicals were contaminated I used the same reagents with another primer and template set and observed no amplification in non - template control.
I think the contamination is in teh primer stocks as they and the template are the only componenets common to both the PCRs. But even after using a different set of primers for the same gene I obtain amplification in the negative control.
Could someone Kindly help me with this problem?

P.S.: In the image the right most lane is the non tempalte control. Kindly ignore the first 2 lanes as they are a different sample.

Nikail Collins
Nikail Collins's picture
primer stock

Hi,When you say you used a different set of primers, did you reconstitute new primers or use a new aliquot?  Make sure you are using a new stock not just an aliquot of the same primer mix.  Otherwise, perhaps check with the manufacturer of the primer to make sure the lot is good.  Also, check to see how they purify the primer.