Troubleshooting problem in PCR for bisulfite Treated DNA - (Jul/28/2005 )

how to troubleshoot the failure of the PCR for bissulfite treate DNA?

There are a number of ways to trouble shoot.

Firstly check that your primers are specifically amplifying converted DNA (ie all non-cpg C's are T's)

Secondly, check that your primer pairs are strand specific ie to one or the complementary strand.

If you can, obtain a positive control, that is to say bisulfite converted DNA that has been shown to work previously. Ideally DNA from the same system and primer sets you are using, if not then just a PCR amplification control being any Bisulfite treated DNA and a separate primer set.

it's best to perform a nested PCR strategy.

Try these out and see how you go.

Good luck!

Nick