oligos phosphorylation with pnk - (Feb/05/2008 )
hi, i have a 12bps oligos. To do ligate, i did phosphorylation at 5 end. i think i faild. i added 20 ul of oligos(100uM), 4ul reaction buffer(10x), 4 ul ATP(final 10mM), water 5ul and 2ul T4-pnk (10unit) . heated @37 for 1hour then heated to 65 for 20 minutes to inactive the kinase. The ATP solution is made by myself by dissolving Adenosine 5-triphosphate disodiun salt powder to TE buffer(8.0).
Does anyone have an idea what i did wrong? Do i need to ajust the Ph of ATP solution?
Thanks a lot. Can you tell me how to ajust and what Ph should it be? Thanks.
we usually adjust the pH to 7. naoh is usually good for the purpose (monitored with a pH meter, but we make large volumes, aliquot and freeze).
Thanks a lot. especially for your quick reply.