other question about BSP - (Nov/19/2003 )

Thanks a lot from pcrman;s advice£¬ ÎÒµÄPCR protocol is same with your saying. I have get the band of control Bisulfit treatment DNA. I think the problem is somthing wrong with my treating DNA process. I¡¡use the protocol according to D.S.Millar etal./ Methods 27&2002& 108 -113 my PCR products band is very weak. I donot know which step is maken a big mistakes. maybe deneature , bisufilte treat , purifiction ?I donot know. gave me some help first thanks cheng

OK, it's clear to me that your problem is the template.

What DNA do you use? DNA from cell line, tissue or paraffin section? What is your pcr product size? Make sure the size should not be bigger than 300 bp although 400 bp was OK at somebody's hands. the best size is around 200 bp.

Don't use too much DNA for modification, use exact 1-2 ug.

Prepare every reagent immediately before use such as NaOH, NaHSO3...
Make sure the first denature step is sufficient ( I use 42C 30')
After adding sodium bisulfite, treat the DNA at 55C for at least 16hr
Loss of DNA during purification is a big concern.

Lastly, try, try and try...

That's what come up to my mind.