Sequencing difficulties - (May/11/2006 )
I am having a lot of trouble obtaining quality sequence from my cloned bisulfite PCR products. The band is of the right size, and gel purified, so I don't think that's the problem. But when I get the sequence back, there's a lot of background noise, and I really only have about 100bp (at most) of readble sequence. I am analyzing a region that is especially GC-rich- does anyone know if these regions, once converted to AT-rich regions during modification, make sequencing difficult? Should the sequencing mix be biased to include more dA/TTPs? I have been having this problem for a while, so any solutions would be helpful.
sequencing from clones should be quite straightforward.
are you using Amersham ET or Big Dye sequencing mix?
Could you upload a chromatogram for us all to see?
A clone should be a pure population of your sequencing vector so you should get very clean sequence, how are you prepping your clones?
If the sequence is GC rich, u might want to consult the perssn incharge of the sequencing as they have special protocol for such problems.
For eg. we had similar problems and our sequencing core ran a special amplification protocol.
You might also want to try using 2-5% DMSO in the amplification reaction.