1 reply to this topic

[ruiiris]

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!

[biznatch]

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.