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.