Bisulfite PCR Question - (Jul/02/2008 )

Hi,

I have trouble in PCR that i can't get product after PCR. I use nest PCR and the cycle is 94 C 3mins, 55 C 3mins, 72 1mins for firest cycle, and 94 C 20sec, 55 C 40sec, 72C 40sec for other 40 cycles, then 72 C 7 mins.
And my product length is 199bp.

This cycle in other CpG islands of me experiment are sucessful, and i had product in one year ago but know i can't have it...

Did anyone have met this problem? or does my cycle have problem in?

Thank you very much for answering my questions.

Hi,

I have trouble in PCR that i can't get product after PCR. I use nest PCR and the cycle is 94 C 3mins, 55 C 3mins, 72 1mins for firest cycle, and 94 C 20sec, 55 C 40sec, 72C 40sec for other 40 cycles, then 72 C 7 mins.
And my product length is 199bp.

This cycle in other CpG islands of me experiment are sucessful, and i had product in one year ago but know i can't have it...

Did anyone have met this problem? or does my cycle have problem in?

Thank you very much for answering my questions.

You could try lowering your extension temperature from 72 to 66, which can help in extending over very high AT regions. What organism is this DNA from?