I am getting tonnes of colonies which I screen by colony PCR and mini-preps and I am just getting a lot of religated vector.
I have tried the following:
A) 1 ug plasmid, digest with 1 ul KpnI-HF with 1 ul CIP for 90 min @ 37 followed by PCR column purification of which 10 ng (based on gel) goes into my ligation.
I first digested for 60 min with KpnI-HF, performed a clean up with a PCR column, then the resulting eluate was incubated with antarctic phosphatase for 60 min at 37, heat inactivated for 5 min at 65 and used 10 ng of this directly in the ligation.
In both cases the insert was digested for 60 min with KpnI-HF, cleaned up with a PCR column prior to ligation. I am using a 2:1 molar ratio of insert to plasmid.
In both cases I am just getting hundreds of religated plasmids. Both my CIP and antarctic phosphatase are fairly new from NEB.
I understand that 3' overhangs are more difficult to dephosphorylate but I really want to crack this problem and finish up the construct.
Any tips for:
-How to reduce the self-religation problem? Using 2 different enzymes for cloning doesn't seem to be an option right now as I'm running out of unique enzymes?
-Any tips to improve success with ligation of large fragments?
Thanks in advance...
Edited by HBImolbiol, 11 February 2013 - 02:54 PM.