Nick translation and ethanol precipitation

Margaret Leversha mal at sanger.ac.uk
Mon Sep 9 07:25:13 EST 1996


Hi all,

I've a few of basic queries about ethanol precipitation of nick 
translation products.  We routinely label DNA for fluorescence in situ 
hybridisation by nick translation using biotin or digoxogenin, then 
precipitate using 1/10 vol NaAc and excess cold ethanol (1ml).  
Depending on the timetable, the tubes are either incubated for 30min 
at -70, or ON at -20.  I've often felt that the second method results 
in a better FISH probe, but that may just be personal bias.

Q.1. Is there likely to be any real difference in the recovery of 
product (c.1ug cosmid DNA to 200-1000bp fragments) with different 
ethanol precipitation methods?  
Q.2. Will the type of hapten have any affect?

After recovering the DNA we add 70% ethanol to the pellet and 
microfuge on max for 10min to remove the excess salts (EDTA etc.).  In 
my previous lab I was always able to pour off the 70% ethanol without 
mishap, whereas now the pellet often partially detaches and can be 
seen as a free-moving bead above a residual adherent pellet.

Q.3. What could cause this change in behaviour?  
Q.4. Is it anything to do with probe fragment length?  

I've swapped to a 80% wash and aspirate the SN but I'm still curious 
about what's going on.

Since my background is cytogenetics, I'd appreciate any pointers to 
references on this subject.

Thanks,

Margaret
-- 
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Dr. Margaret A. Leversha              |        
Molecular Cytogenetics                |         
The Sanger Centre                     |
Wellcome Trust Genome Campus                   |
Hinxton                               |
Cambridge                             |        Tel. +44 (0)1223 494800  
UK      CB10 1SA                      |        FAX  +44 (0)1223 494919 
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