my negative PCR control is still positive ! - (Apr/25/2008 )
I had a similar problem back in January and it caused me to lose a lot of sleep! After trying everything (replacing all reagents) I stumbled across a paper on fungal and E.coli DNA contamination in Taq preparation. I switched from NEB taq (very cheap) to Amplitaq gold LD (low DNA) and haven't had the problem since! I could only conclude that I had been amplifying Elongation factor from either E.coli or a fungal contaminate in the taq!! So next time your supervisor suggests using half priced enzymes, reconsider!
I have been struggling with a contamination problem for 2 weeks now. I tried changing the water, the reagents and the primers but I still have bands in my negative control. When my lab mate tried with my reagents and her primers, the bands did not show up. I tried with her primers and my new primers but they still appeared in my negative control on my gel. I do not see any difference in our techniques, or the way we make our PCR master mix. The only thing I have not tried until then was changing my PCR tubes. I autoclaved my new PCR tubes and we both tried doing PCR with same the reagents and primers. Negative control bands did not show up. We thought our problem was solved but after two days they started appearing in my and her negative control samples. So now we both have the problem. The weird thing is they are not same for all my runs. They are changing from one run to another run. Sometimes I can see only one band that is further from my 100bp marker and after the dye mark (I guess it is a primer band) and sometimes there are multiple bands with my primer band. From these results, I can say there is something affecting my PCRs in an off and on fashion. After reading all your suggestions from this forum, I have cleaned all my stuff with bleach and tried PCR. Still there were bands in my negative control. The only thing I did not try was cleaning the internal tubes of my pipetters. I am planning to try that tomorrow. In the meantime, if I can get any suggestions from you, that would be really helpful for me.
Thank you all !!
ok...as I really need to finish this project, I am using shrimp nuclease. I treat my PCR mix before adding the DNA template. I also treat the water I use for diluting the DNA samples. For that, I mix water, Tris-HCl, MgCl2 and the nuclease (2units).
The PCR negative controls now come out negative but extra peaks are still present int he samples. What I mean by extra peaks is that all 4 alleles are present. I use GeneMapper to call the alleles. In nearly all the samples, only 2 alleles (out of 4) are called by the software. The other 2 peaks are not called, even if they are as strong as the called ones. I don't know if I can really trust these results.
I am now wondering if the contamination could be present in the DNA samples themselves. Maybe they got contaminated during the extraction step, ie by a dirty pipette ?????