PCR smears are getting me crazy! - (Feb/17/2006 )
I designed a pair of primers 5 months ago and they worked well all the time. I was getting bright bands till 2 weeks ago, and then I got suddenly smears in all my samples. I’ve change DNA template, all reactive for new ones (MgCl2, Buffer, Taq, dNTPs...), different water, even I asked for new primers twice, I’ve change pipettes, laboratory, and Termocicler. Everything point to there’s no contamination anywhere because everything was new and I performed it in a different laboratory but I got the same smear. So... should it be the primers? What could be wrong with them? Dimmers? Why now and no before during the 5 months I was using them? Could it be because of any change in the synthesis process of the company? If I ask the primers to another company, will I have to change the conditions for the PCR? I prefer not to change the conditions, because I used them for several experiments, and it would mean that I have to repeat all of them? .
Has someone any idea?
Thanks a lot!
What are your rxn conditions? What's the volume of your rxn, type of template, etc.? This sounds like template overload.