Ligation of double insert into pET31 failing - (Nov/24/2011 )

Hello,

I would appreaciate any advice here, it feels like I've been banging my head against a brick wall for weeks now.

I am trying to clone an 81 bp fragment (in two halves with a 4 bp overlap) into pET31, however after ligation and transformation I get ZERO colonies.

I digest pET31 with AlwNI, dephosphorylate and then gel extract the linearised vector. This is fine. After this however I can't get my insert to anneal. I anneal the two halves separately and then ligate all together with the vector in the same reaction. I have tried ligation overnight at 4', on the bench for 2h at room temp, and have altered most of the parameters I.e. reduce the amount of ligation mixture (either 5 or 10 uL) per 60 uL of competent cells.

Does anyone have any ideas?

Thanks

Do your oligos have a 5' phosphate? If not, that would explain the problem. You can add them with a PNK treatment before or after annealing.