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Problem of Direct bisulfite sequencing

Bisulfite direct sequencing

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#1 ruiiris

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Posted 16 May 2012 - 01:30 PM

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!

#2 biznatch

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Posted 28 May 2012 - 04:38 PM

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.





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