5'-end labeling of 8mer oligo?

Uttam Rajbhandary bhandary at wccf.mit.edu
Mon Nov 14 17:27:00 EST 1994


In article <9411120046.AA20356 at pollux.cc.umanitoba.ca>, pzhang at cc.UManitoba.CA (pzhang) writes...
>Hi, netters,
>I posted my broblem a couple of days ago. Having got no reply, I'd like to try again.
>I want to radioactivly label 5'-end of a 8mer oligo. I am not sure how to separate labled oligo from the reaction (removal of ATP, ADP etc.) I want to use this oligo as a strand of an adapter. So, my second question is: If I use the labeled reaction dire
> 
>Any ideas and suggestions are highly appreciated.
> 
>Peijun Zhang
>pzhang at cc.umanitoba.ca


I would use either DEAE or PEI cellulose, developing in 0.75M KH2PO4.  The ATP 
will elute, and the oligo will stay at the origin, or move a bit off it.  You 
can cut out the spot with your labeled oligo, and elute with a high 
concentration of some volitile salt buffer, either TEAB carbonate, or ammonium 
acetate.

An alternate approach is to use a Waters Sep-Pak column.  Bind your sample at 
low salt.  Wash with 50mM TEAB carbonate to get rid of excess ATP, etc., and 
then elute your oligo with 60% methanol.  dry down, and dissolved in water or 
the buffer of your choice.

Harold Drabkin




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