PCR amplification in negative control - (Mar/29/2001 )
I have been troubled and been nearly ripping my hair out with this PCR problem. I always run PCR samples with negative controls which include all the reagents including the primers but minus any template. The last two PCR sets that I have done have come out negative with regards to the reactions with DNA templates in them (8 samples) while the negative controls have turned up positive and with the desired band range. I have used the Hot Star polymerse kit for the PCR reactions and the samples were run using temperature gradient PCR machine. 3 sets at various temperatures of the samples were done. The DNA template samples used were about a year old and frozen. I would welcome any reasoning behind this weird PCR experience. I understand if I got results in my samples and the negative controls meaning definite contamination but the fact that I see products in the negative controls and not my sample reactions stumps me.
If I were you, I would discard everything (water, PCR reagents, primers, tips)except the template. Comtamination is definitely the problem.