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

W.E.Corver@lumc.nl W.E.Corver at lumc.nl
Wed Jul 15 12:39:50 EDT 2009


Dear Tomas,

Our experience is that PI is mostly removed along the way during sample
work up and DNA purification.
First the sorted cells are spun down and digested, frequently by prot.
K. Then the sample is boiled (to inactivate prot. K), spun down and DNA
in the supernatant is further purified (including precipitation/binding
and washing steps). After PCR traces of PI will be further diluted. We
do a lot of DNA content sorting, especialy for detection of chromosomal
aberrations using fluorescent dyes. We use al kinds of controls. Data
are excelent and are confirmed by other techniques on the same material.


Kind regards,

Willem Corver 


Willem E. Corver, PhD
 
Dept. of Pathology
Leiden University Medical Centre
P.O. Box 9600, Building 1, L1-Q
2300 RC  Leiden
The Netherlands
 
Tel: +31 71 526 6580
FAX: +31 71 524 8158

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Darzynkiewicz,
Zbigniew
Sent: woensdag 15 juli 2009 17:20
To: 'Tomas Adejumo'; Cytometry Mailing List
Subject: Re: [Cytometry] Is it possible to purify DNA once stained with
PI?

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


-----WIBR-EMAIL-----
This email and any files transmitted with it are confidential and
intended solely for the use of the individual or entity to whom they are
addressed. If you have received this email in error please notify the
system management (it.support at wibr.ucl.ac.uk).
--------***---------

_______________________________________________
Cytometry mailing list
Cytometry at lists.purdue.edu
https://lists.purdue.edu/mailman/listinfo/cytometry
_______________________________________________
Cytometry mailing list
Cytometry at lists.purdue.edu
https://lists.purdue.edu/mailman/listinfo/cytometry


More information about the Cytometry mailing list