Do NTP's precipitate with ethanol? How efficiently?

Robert Slany rslany at leland.stanford.edu
Sun Feb 11 00:39:22 EST 1996


CK Wen wrote:
> 
> Dave Hollingworth <d-hollin at nimr.mrc.ac.uk> wrote:
> >If you do an ethanol precipitation on a mixture of NTP's and DNA
> >do they both go into the pellet? Do the conditions for precipitation
> >matter (different salts, temperatures etc....). Is there a better way
> >of precipitating NTP's?
> >Dave
> >
> Dear Dave:
>   I do not think dNTPs precipitate with the DNA, though no direct
> evidence can be provided to you.  Just think that when we prepare
> P32-end-labeled DNA probes, the gamma-P32-ATP is removed after ethanol
> precipitation.  Also, if you EtOH ppt your alpha-P32-lableled DNA
> probes, you may get "hot" sup which indicates that the free dCTP is in
> the EtOH phase.  After several EtOH washes, the sup is never hot and the
> pellet should be the labeled DNA.
> 
>   Hope this help.
> 
> Cheer,
> 
> Chi-KuangHi Chi-Kuang,

from my personal experience I can't agree with your statement. Although I do 
believe that the efficiency is very, very low in an experiment done by myself 
not only enough dNTPs but also Klenow enzyme survived ethanol precipitation 
to fill in the ends of the following restriction digest. 
The morale: don't save time by omitting heat inactivation. It took me a week 
to figure out that the cloning was actually blunt ended!
Also in vitro transcribed and subsequently precipitated RNA contains tons of 
nucleotides that have to be removed by chromatographic steps. 
If you need it absolutely nucleotide free, do a DEAE-Sepharose purification.

Just my 0.02$ 
Robert
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