HMW band in well after bisulfite PCR - (Sep/06/2006 )

Hi everyone,

For months, I had been successfully amplifying a 500 bp region of a gene after bisulfite treatment using 2 rounds of semi-nested PCR. Usually I would set up 5-10 rxns of the 2nd round and pool them together (EtOh precipitated them together) to ensure I had enough product for subsequent cloning. Then, I would digest them overnight with the appropriate restriction enzymes, run the digest on a gel, and gel-purify the resulting band for cloning.

I have run into the problem now of running out the EtOh precipitated PCR products on a 1-2% agarose gel and getting a high molecular weight band in the well that doesn't really migrate or a HMW smear. I'm using the same primers, same conditions, have tried new reagents (in case the old ones were contaminated), used different preps of genomic DNA. The only thing I can think of is that the restriction enzyme digestion was necessary before running out on the gel in order to visualize the product? That maybe i have a lot of primer dimer/junk DNA that has to be digested into small pieces for the amplicon to be visualized?

if anyone has any thoughts on this, they'd be much appreciated! this has been so frustrating the last few months!

Thanks,
K

sounds like genomic DNA contamination but it may not be in your PCR, could your gel combs be contaminated with something tht fluoresces with EtBr? i have found that previously.

have your tried reording your primers and repeaating the experiment? you primers could be contaminated.

N