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2nd round of amplification issues - (Jun/07/2006 )

Not sure what to make of this. I'm amplifying a 650 bp product out of a fairly small amount of genomic DNA for sequencing. The problem I'm experiencing is that using a single round of amplification ~30 cycles I dont obtain enough product to visualize on a gel. So I tried to use the same primers and perform a second round of amplification, but instead of a band I obtained a huge smear. Oddly, when I take the first round product and try to amplify using my sequencing primers, I get a beautiful band that is exactly the size I wanted with no smearing or spirous bands. Is smearing a typical result from using the same primers in multiple pcr rounds?

I've actually thought about taking the product that I amplified using my sequencing primers and getting that sequenced with the same primer set, but was told that I'd likely run into sequencing problems and that sequencing primers should idealy not have to bind directly to the ends of the template. Does anyone have experience with this?


I suggest you to take the first reaction and to dilute it at least 1:100 and then use it for the second reaction. In addition you may try to do the second reaction with gradient temperatures.