[Cytometry] Is it possible to purify DNA once stained with PI?

Darzynkiewicz, Zbigniew Z_DARZYNKIEWICZ at NYMC.EDU
Wed Jul 15 11:20:17 EDT 2009


Hello Tomas,
The intercalating dyes such as PI, EB acridine analogues can be removed from DNA by caffeine. Exposure of PI-stained cells or PI-stained bands in gels to 0.05M solution of caffeine in PBS effectively removes the dye from nuclei of the stained cells or from the DNA bands in gels (see; Bedner et al.,  Caffeine dissociates complexes between DNA and intercalating dyes. Application for bleaching fluorochrome-stained cells for their subsequent re-staining and analysis by laser scanning cytometry. Cytometry 2001; 43:38-45. http://www3.interscience.wiley.com/cgi-bin/fulltext/76502815/PDFSTART
Dialysis of the extracted PI-DNA (contained in the dialysis tubing) against such solution of caffeine should purify DNA. Caffeine has relatively high binding efficiency to intercalating dyes/drugs by forming heterologous stacking complexes and thereby removes PI from the intercalating complexes by the competitive binding.

Zbigniew Darzynkiewicz, M.D., Ph.D.

Professor of Pathology and Medicine
Director, Brander Cancer Research Institute
New York Medical College
BSB, Room 438
Valhalla, N.Y. 10595

www.darzynkiewicz.com/zbigniew/


-----Original Message-----
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Tomas Adejumo
Sent: Wednesday, July 15, 2009 9:54 AM
To: Cytometry Mailing List
Subject: [Cytometry] Is it possible to purify DNA once stained with PI?

Dear All,

A colleague wishes to sort cells based on DNA content and analyze using
PCR. Would the bound PI affect downstream analysis or is it possible to
purify?

Many thanks, Tomas

Tomas Adejumo MSc
Flow Cytometry and Confocal Microscopy Manager
Scientific Support Services..."Bringing ease to research.."

Scientific Support Services
Wolfson Institute For Biomedical Research And UCL Cancer Institute
The Cruciform Building
UCL
Gower Street
WC1E 6BT

Tel: 0207-679-6719

http://www.ucl.ac.uk/wibr/services/index.htm


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