Problem of Direct bisulfite sequencing - (May/16/2012 )

Hi everybody,

This is my first time to do methylation study. I'm now trying to do direct bisulfte sequencing. I'm using the Imprint DNA modification Kit from sigma and I do 2 rounds of PCR (including one semi-nested PCR). When checking from the agarose gel, I saw my desired band pretty nicely except that I have another weak unspecific band underneath. I tried to sequence it directly, but I got a high background which is full of "C" signal everywhere from the forward primer. Then I tried to gel extracted my desired band as I suspected the background was caused by the underneath unspecific band. However, I got similar highly noisy result which is impossible to read through.

Dose anybody have any idea about this?

Many thanks!

Maybe I'm missing something, but aren't you supposed to clone the PCR product before sequencing? When I did this I gel purified, cloned, then sequenced a bunch of clones.