Primer contamination - (Nov/07/2001 )
Hi Is there anyway to get rid of primer contamination? I have a band in neg control that correspondes to the size of micrsatellites I am working on.Thanks
Isn't it template contamination? I always thought that the best negative control for PCR was to have a no template tube with you primers, taq, dntp's and buffer. I think that as far as PCR goes, it takes a lot more primer than template to amplify something. With that said, even though my lab uses primers but no template in our negative controls, we still have the same problem you have. Try setting up your reactions under a hood, or in an enclosed, sterile area. Also, try wiping down the inside of your thermocycler and start over with everything fresh. (PCR mix, etc.) If you are sure it is primer contamination, then you might look into getting some type of nucleic acid purification spin columns. Pharmacia is a good place to look. I believe there are some columns that exclude nucleic acids that are smaller than 100bp for example.
Try pharmacia's microspin S-400 HR columns. They are meant to puify PCR product away from unincorporated primers (< 32mers). Try running the sample you use for your negative control through these before PCR. Good luck!