Nick translation and ethanol precipitation

Margaret Leversha mal at
Mon Sep 9 07:24:47 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.


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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