cloning a 3kb fragment into a 6kb vector - or maybe an EcoRI/HindIII double digest problem? (Oct/12/2007 )
Hello all,
We're having problem to clone a 3kb frament into a 6kb vector. It comes from a PCR amplification, digested by EcoRI and HindIII. The vector was also digested with these enzymes, CIP treated and gel purified. We tried different insert/vector ratio for the ligation, but never get any clones. We also prepped the vector backbone a couple of time (this is usually the critical step in my hands).
I can't really think of any thing to change except to do single digests, but the NEB catalog says that it works in the EcoRI buffer, and I usually trust the NEB catalog.
Does anyone have any suggestion? Thank you so much!
I'd stop CIP treating your vector. For a double digest you should have low background in any case. CIP treatment makes cloning more difficult in the best of cases, and makes it impossible in many due to damage to the ends.
We're having problem to clone a 3kb frament into a 6kb vector. It comes from a PCR amplification, digested by EcoRI and HindIII. The vector was also digested with these enzymes, CIP treated and gel purified. We tried different insert/vector ratio for the ligation, but never get any clones. We also prepped the vector backbone a couple of time (this is usually the critical step in my hands).
I can't really think of any thing to change except to do single digests, but the NEB catalog says that it works in the EcoRI buffer, and I usually trust the NEB catalog.
Does anyone have any suggestion? Thank you so much!
You donot need CIP treatment for sticky end cloning. Try the ligation without CIP treatment of DNA, I guess it would work.
Good Luck !!!
well as already mentioned CIP is probably the cause of your woe. Over dephosphorylation is a real problem. Too much CIP for too long will damage your vector's overhangs. This renders the vector unligatable. Listing you dephosphorylation conditions and how much DNA you are dephosphorylating would help make certain this conclusion.
And as already mentioned, since your ends are non compatible, vector religation is not a major problem.