5'-end labeling of 8mer oligo?

Peter Gegenheimer peterg at rnaworld.bio.ukans.edu
Tue Nov 15 17:43:12 EST 1994


In <3a8h85$7oq at senator-bedfellow.MIT.EDU>, barry at aaRS (Barry Henderson) writes:
>pzhang (pzhang at cc.UManitoba.CA) wrote:
>: 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 di
rectly( without any purification) to go ahead, what are the ligation results?
>
>: Any ideas and suggestions are highly appreciated.
>
>
>Why not just EtOH precipitate it and use it directly.  Your precipitation
>efficiency may be a little low with such a short oligo but you should
>be able to get enough back to work with.  Alternatively, gel purify it.
>I routinely purify RNA oligos between 7 and 35 nts long on high percentage
>(16%) denaturing acrylamide gels then extract the band you want (visualized
>by UV shadowing or if it is labeled by autoradiography).
>
>Barry Henderson

EtOH precipitation in the presence of Mg(2+) will also ppt. the ATP. For most 
purposes, the kinase reaction mixture can be usesd directly in subsequent reactions. 
You con use any standard kinase reaction buffer, or try the buffer to be used for the
subsequent step. T4 PNK likes Tris, Hepes, or even Pi buffer, pH 7 to 9; some Mg; if 
you use equimolar [ATP] to [oligo] you'll get very efficient (70-100%) utilization of
ATP.

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