precipitating nucleotides protocol

Mircea Podar podar at utsw.swmed.edu
Tue Mar 18 17:14:57 EST 1997


In article <rollins.24.9.332C1EC2 at postbox.acs.ohio-state.edu>,
rollins.24 at postbox.acs.ohio-state.edu (Sean Rollins) wrote:

>  I'm trying to synthesize 4 thio UTP and I'm losing my sample at the 
> precipitation step.  The protocol I have contains 20mM ATP and 10mM
> 4 thio UDP in 100ul and the phosphate is transfered.  This is brought up to 
> 500ul with 300mM sodium acetate and 0.1% SDS and precipitated with an equal 
> volume of 1:1 ethanol/acetone.  I've checked my reaction by HPLC and I'm 
> losing my sample at the precipitation step.  No details are given on this 
> precipitation step (time, temp and spin conditions).  If anyone has any
advice 
> or any 4 thio UTP for a possitive control it would be appreciated.
>                                 Thank you 
>                                 Sean Rollins
>                 Rollins.24 at osu.edu



Hi Sean,

I synthesized that stuff some years ago, and I also used 1:1 etoh-acetone.
For time reasons, I left it o/n at -80 C to precipitate. Spin it in a
standard microfuge (15k) for 15 min at 4 C. From my experience with small
oligos , you can actually add 2-3 volumes of the ethanol-acetone and will
help on recovery. If you want to experiment with your precipitation
technique without losing the important stuff, put a bit of hot NTP in a
mixture resembling your reeaction and see if you get good counts getting
down.
Are you going to put the crude 4sUTP on the DE 52 column? That worked fine
for me, and the final lyophilization was OK also. If you keep your final
product at -80 C it will last you for years. Good luck,

Mircea Podar
U.T.Southwestern Med. Ctr. Dallas
podar at utsw.swmed.edu



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