Posted 24 September 2003 - 04:13 PM
You think that's bad! I get products of the 1st round and 2nd round, AND cross amplification between the 1st and 2nd round primers, so I usually get 4 bands! Without adding an extra cleanup step (via exo/SAP or a spin kit etc.), which of course adds to the risk of contamination, we've come to the conclusion that it's just an artifact of nested PCR's. It can be reduced by reducing the concentration of the primers in the first step or as the last person said, reducing the amount of 1st round reaction you carry over to the 2nd, BUT this will reduce the sensitivity of the whole process and you may not pick up the band you want, particularly if the copy number in the original template is low, as is the case with us.
If you do figure out a way to deal with it you're better than us, please let me know.