DNA/RNA ethanol precipitation

Stephen R. Lasky, Ph.D. Stephen_Lasky at brown.edu
Fri Jun 2 08:49:13 EST 1995


In article <drm21-0106952054510001 at drm-mac1.welc.cam.ac.uk>,
drm21 at mole.bio.cam.ac.uk (David Micklem) wrote:


> My recollection is that there was an article (BRL Focus?) that looked at
> this quite a long time ago.  If anyone has the exact ref, I'd like to see
> it again.
> 
> Anyway, so far as I remember, the conclusion was that chilling (at all)
> was unnecessary, and that the longer the spin the greater the recovery. 
> For dilute DNA, addition of carrier (I prefer glycogen or linear
> acrylamide) helps a lot.

Actually , were two articles on it.  One was in Focus, Fall 1985.  And I
think that the conclusion in that article was that only two things
mattered in DNA ppt (at 0.3 M NaAc, ph unadjusted).  The first was the
concentration of the DNA and the second was the legnth of centrifugation: 
The more concentrated the better and you need to centrifuge at least 20
minutes, but 30 is better at lower DNA concentrations.  Incubation time
didn't matter.

A later issue, spring 1987, comparing the effieciency of ppt with NH40Ac
vs NaAc also looked at time, temp, and concentration:   That article
showed that with NH40Ac, recoveries of concentrated DNA solutions were
best at 0 decrees C or at room temp (best at RT) no matter how long you
incubated (0 hours to o/n), but that at lower concentrations (0.005 ug/ml)
recoveries were best with and overnight incubation at room temp or at 0
degrees C.  Use of -70 deg C or even -20 decreased the yield at all
temps.  The 

Take home point from both articles is that if you have dilute DNA
solutions that are free of nuclease activity, you should incubate
overnight on your benchtop (or in the refer if you are paranoid (I am)). 
If you have concentrated solutions (> 5 ug/ml) , add your salt (final
concentrations: 0.3 M NaAc (I would adjust the pH to 5 to 5.5), or 2.5 M
NH4OAc (pH unadjusted)) mix (contrary to the comments of one of the
responders, I vortex most plasmid preps and don't see any shearing.  These
things are tight little balls after you add salt and ethanol, and are
usually small supercoils to begin with, so are less susceptible to
shearing than the long rods you have in genomic DNA) and spin at room temp
for 30 minutes (recovery goes down with lesser times).  I usually use a
chilled microfuge because mine tend to heat up on the benchtop.

IMHO it seems like it might make sense to ppt in isopropanol since it
requires less salt and less time, therefore making the above moot.

Just reporting the jist of the articles and adding my $0.02. 8-)

SRLasky

********************************************************************
Stephen R. Lasky Ph.D.  Brown University/Roger Williams Medical Center
Landline: 401-456-6572   Fax: 401-456-6569  E-Mail: Stephen_Lasky at brown.edu
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
America may be unique in being a country which has leapt from barbarism to decadence without touching civilization.  John O'hara
********************************************************************



More information about the Methods mailing list