So I have a plasmid that contains an insert with NdeI and BamHI into pet3a. My aim is to do an N-terminal deletion. So I have designed a Forward primer with NdeI site and a reverse primer with BamHI site. The BamHI has AAAAAA as extra and NdeI has AAAA as extra. The two have good compatibility and the PCR reaction goes well, where I get the right sized PCR product as my insert.
To get my vector, I cut the original plasmid with NdeI and BamHI double digest, which doesn't go to completion, but I gel extract and pool 5 digestion reactions.
I then also double digest the PCR product (I have also tried sequential digests) and purify it with qiagen kit.
In my ligation, i put few varying ratios of insert vs. vector and do an overnight 16C ligation. I heat inactivate the ligase and use 5 uL of the reactions for transformations into BL21 electrocompetent cells. The transformation efficiency is good as my control (the initial plasmid) seems to produce a near lawn. However, I don't get any colonies for my actual trials.
What could be the problem?
I tried to pinpoint the problem by running my ligations on a gel but I guess you can't really judge anything from that..
Really would love some help. Thanks for your time.