PCR contamination in negative control - (Jun/30/2004 )
I have been contamination problems in my no DNA control of PCR. I have changed all the solutions and have got new primers made. But some how this contaminating band always appears. This happens with a particular DNA clone and not with other clones. Also there is one particular seuence of primers which doesn't give me contamination with this clone. I set up the reactions in a hood. I don't know what to do?
Two sources of contamination that produce a band in the No-DNA control are:
1. Mycoplasma contamination with a genome GC strength of 27.5%- it is everywhere; including the HEPA filter of your hood.
2. Your primers could carry linear adducts of the molecule resulting in lengths upto 250 nucleotides. This happens due to condensation reactions that were not arrested at the right time during thesynthesis. If you ordered new primers but did not change the manufacturing company, maybe you should. Run a sephacryl S-300 gel filtration on your primer batch and check for more than one 260 nm absorbing fraction.
Thanks for your suggestions. Do you think primer purification or mycoplasma contamination could be a problem even if the band is of the same size as the product expected in positive control (with DNA)? I am trying to amplify a prokaryotic vector sequence integrated into mouse genome. My primers are from qiagen. One particular set of primers works well with this construct.
have your solved your problem yet...i group member had the same problem and it worked out that the gloves was the source of contamination