Facsing isolated DNA

From: Michael Ormerod (Michael_Ormerod@compuserve.com)
Date: Mon Jan 15 2001 - 16:48:54 EST


Message text written by Connie
>

I have someone here at Columbia who wants to do a two steps staining for a
DNA adduct with a 1' monoclonal antibody and a 2' FITC conjugated antibody.
The major problem we have is that they have to isolate the nucleus from
cells with a nuclear isolation buffer, then treat the isolated nucleus with
2N HCL to unfold the DNA in order to expose the adduct.  At last, they do
the staining to look for presence of the adduct in those HCL treated
nucleus
(the nucleus most likely are lysed already with only DNA remaining).
Does anyone have any recommendation in doing FACS with this type of DNA
staining (FACSing only DNA, that is not confined within a nucleus/cells).
I
would expect the size of the DNA to be smaller than debris, so what should
I
do with the FSC/SSC.  And, how should I interpret the data, since I don't
even know whether I am looking at debris or DNA.<

I would try fixing the nuclei first in ethanol, then opening up th Edna
with 2 N HCl. This procedure works OK when staining nuclei for BrdUrd.

Alternatively, why cannot you use whole cells fixed in ethanol? Again this
works OK for BrdUrd.

Michael Ormerod
34 Wray Park Road
Reigate RH2 ODE
Telephone: +44 (0)1737 241726
FAX: +44 (0)1737 226736
Mobile telephone: 07802 293242



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