Protocol Online logo
Top : New Forum Archives (2009-): : General Lab Techniques

Nuclease contamination or what??? - (Sep/01/2009 )

Hello all,

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

-nicksailor-

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.

-phage434-

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 :P). Sounds like your PCRs just arenīt working. Make new dilutions of the primers, and use a fresh aliqote of dNTP....

-Chimp-