Bisulfite Treatment

Alan Smith alansmith at students.wisc.edu
Fri Jul 27 15:40:09 EST 2001


Hey Kelvin,

I have the same trouble with bisulfite treatment.  I think the high salt
concentration causes problems with ppt of DNA.  I usually treat 10ug of DNA
and get back a lot lot less and it is badly degraded.  With this in mind i
never try for PCR products larger than 500 bps.  If I keep the product small
I don't have a problem even if i cant see the treated DNA on a gel after
cleanup.  Be sure and run good controls.  Bisulfite PCR has many problems in
my opinion.  Specifically you are going to amplify a Methylation state that
may only be present in a small group of cells.  Be sure and do southerns
first to confirm the Methylation levels in your primer target area.  Daphne
Pruce at the University of Chicago has been doing some good work looking at
the methylation levels around centromeres in Arabidopsis.  Check out some of
her papers and we use the bisulfite treatment method from this paper: Urea
improves efficiency of bisulphite mediated sequencing of 5-methylcytosine in
genomic DNA (1998) Paulin et al.  Nucleic Acids Research 26 5009-5010.

Good Luck

Alan Smith

-----Original Message-----
From: owner-methods at hgmp.mrc.ac.uk
[mailto:owner-methods at hgmp.mrc.ac.uk]On Behalf Of Kelvin Joe
Sent: Friday, July 27, 2001 6:10 AM
To: methods at hgmp.mrc.ac.uk
Subject: Bisulfite Treatment


I try to investigate the methylation pattern of cancer. When I did bisulfite
treatment, I couldn't get enough DNA to do downsteam PCR. The problem might
be DNA degrade when incubated with sodium bisulfite.
Does anybody have the experience of bisulfite treatment? If you do, please
help me.

Thanks a lot.

Kelvin


<http://www.biowww.net/forum/read.php?f=1&i=3165&t=3165>

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