Nuclease contamination or what??? - (Sep/02/2009 )
I have this huge problem going in the lab. I am a newbie in molecular biology, but have been working in microbiology for last 2-3 years or so. So here is the problem: I am trying to PCR amplify the linear DNA and transform it in the bacteria. Evergything was working fine before, but one day all of a sudden everything just stopped working. When I amplify the DNA and run on the gel there is just NOTHING when I look at it under the imager. none of my PCR reaction work, the gel is just clear but there is a HUGE round smear at the very end of gel, which does not correspond to any possible size of DNA (side question - can EtBr bind to nucleotides?). I tried autoclaving the pipettes for 20min dry cycle at 121C, changing the dNTP-s, using the fresh water, changing gloves, changing the running buffer, and after all these stuff, I got some bands that are right size, however its really faint and there is still that huge smear at the very bottom of the gel. my lab-mate, who is working the next bench did the SAME PCR amplification but with own equipment and samples and his looks fine.. I just dont know what to do, what is my mistake and how can I correct it. It has been preventing me to complete my simple experiments for 2 weeks already and its getting SO frustrating. Please if anybody has any suggestion, do share it.. Thanks
Which end of the gel is showing a smear? If it is near the well, this could be very large amounts of genomic DNA. You may be using far too much (100-1000x) template in your reactions. Try a 100x dilution. If you still have problems, switch to your partner's reagents and machine and see if the problem persists, or have him/her run one of your reactions.
If it is on the other end of the gel it might be primerdimers (if you run the gel long enough they get to the very end ). Sounds like your PCRs just arenīt working. Make new dilutions of the primers, and use a fresh aliqote of dNTP....