Standard PCR banding? - multiple bands instead of one (Sep/13/2006 )
I have started to encounter multiple bands present instead of correct sized single amplicons. I am amplifying a 790bp product from the 16S rRNA gene using the same reagents successfully used for the last month and all of a sudden multiple bands generally +/- 200bp are appearing either side of the target amplicon size. The only new factor involved is the water, which I treat with Diethylpyrocarbonate the same way I have the last 8 years with no concern. Appart from this everything else is the same. I perform approx 6-8 of these PCR's a week and they have only started yesterday and again today. Even the positive control is splitting into 3 distinct bands!!!
Any help appreciated
Have you done a negative control? All the reagents but no template. It sounds like maybe there's some contamination somewhere.
I run two negs per reaction, a no-DNA template and a no primer template, both are fine. This phenomenon only affects my DNA template lanes, including the Pos control which was working fine last week??
perhaps order a new set of primers? your primers could be degrading...how do you store them?
Primers stored at -20 in 100ul working aliquots. Just seems funny how they turned ugly almost overnight? I'm currently running another PCR using the identical reagents as well as another parrallel PCR using fresh primers and dNTP's. Will keep you's posted.