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PCR help - (Apr/18/2007 )

Hi all,

I need help. I have been running PCR on a double transgenic line and have constant issues with determining negative and postiive results. For example, when I first started the PCR for the 1st transgene there was either a band or there wasn't, the second trangene was the same. After about 6 months, for the first transgene I started to notice a double band in the "negative" animals which matched with my negative control animal, so I could still differentiate a positive from a negative animal. THis past month the bands have become more intense and the same size as the postive, but they vary in intensity and are sometime a little less intense than the positive and sometimes almost completely clean. Generally I have multiple intensity bands of the same size in a single gel. At the same time, I ran another PCR with the second transgene and it was fine...either a clear band or no band. I used the same DNA samples, same water, same taq, etc. so I know my components aren't contaminated. I figured my primer for the first trangene may have been degraded or contaminated and made a new aliquot from the original stock (which was used to make the first aliquot of primers that used to work and has not been touched since that orignial aliquot was made), but this failed to produce different results. I'm stuck. How is it possible that this might be contamination??? Any other ideas for why this has suddenly stopped working?



It sounds like you may have something floating around in your lab... especially because of the variance in whether it is completely clean or not. do like UV sterilization change your lab coat etc (see other posts from bioforum on PCR contamination for other ideas) This is what it sounds like to me...

Good luck!