Nucleotide precipitation protocol

Sean Rollins rollins.24 at postbox.acs.ohio-state.edu
Sat Mar 15 09:55:50 EST 1997


        I'm trying to synthesize 4 thio UTP, but I'm losing my final product (I think 
at the precipitation step).  I can see my 4 thio UDP peak shift on HPLC 
directly after my reaction and after phenol extraction.  My existing protocol 
calls for me to bring the volume of my reaction upto 500ul with 300mM 
NaOAcetate and 0.1% SDS and then precipitate with 1 vol. 1:1 ethanol/acetone 
(by the way my initial reaction contains 20mM ATP and 10 mM 4 thio UDP in 100 
ul). I'm not much of a biochemist and no details were given in the protocol 
for the precipitation step eg. temp, time, spin speed or length of spin.  If 
anyone has any suggestions or if anyone has any 4 thio UTP as a positive 
control it would be greatly appreciated.
                                Thank you
                                Sean Rollins
Rollins.24 at osu.edu




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