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Drop down during bisulfite sequencing - (Mar/12/2011 )


As a new person in this field I did some reading in this forum.
I'm facing a problem with my bisulfite samples during sequencing. Unfortunately I couldn't find anything. I didn't read all the topics since there are many.

I do have a problem and I hope some of you can give me some help/advice.
The problem we are facing is during the sequence reaction of our clone with bisulfite treated DNA.
The reaction drops-down after 10-20 bp of the insert.

We are using a pBluescript plasmid which we made linear ourselfs. All the steps (gDNA isolation, bisulfite treatment, PCR) goods fine.
Except the sequencing.
For the plasmid isolation we use a miniprep kit of Fermentas. We also did an extra purification with phenol/chloroform. This didn't had an effect on the sequence reaction.
The sequence-primer we used is a M13 forward. We also tried other primers....but nothing gave a better results
Is it a vector problem? (When we do a cloning with no bisulfite samples the sequence reaction goes drop-down)

I hope someone can help me.

Thanks in advance!



We've never had similar problem sequencing cloned bisulfite PCR products. We use commercial service providers and they process our samples by standard procedures without even knowing our samples contain insert amplified from bisulfite treated DNA. We use stand vector primers for the sequencing reaction. I think the problem is with your plasmid. Have you verified the insert by restriction digestion? Make sure your plasmid is from single clones and the preps are clean, the amount of DNA used is optimized. In short, there is nothing special with cloning and sequencing bisulfite PCR products.



Thanks for your replay.
We also think that the plasmid is giving the problem. But we don't understand why.
All the advice you are given us is what we already do....that isn't the problem.

We will keep on trying!