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?
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