I have a multi-domain recombinant protein I'm trying to clone. So let's say I have plasmid-gene pAB and I want to cut out B and put in C, gaining plasmid pAC.
To do this, I have been cutting out B with the restriction enzymes MfeI-HF and PstI-HF, as well as the restriction enzyme BsaBI in order to further cut B in half. I then cut out my backbone and purify it. As for C, it is a free oligo, so that I have just cut and then purified it without running it on a gel. The last time I attempted this, I further used antarctic phosphatase on my backbone (pA) to prevent it from closing on itself or reincorporated any of the original insert ( B ) that made it through. I then ligated my pA with C in a 1:6 molar ratio, inactivated it, and transformed cells.
Here's the kicker that is showing up repeatedly: my control plate, with only the backbone, will have 0-3 colonies. My ligation plate will have 100-200. And yet restriction analysis (with HincII and AgeI-HF) vastly shows the damn original insert.
What the hell could be going on? I'm about to test my intended insert and the primers I'm using to amplify it to rule out contamination by the unintended insert, but that would be very surprising.
Do y'all have any other ideas?
Edited by RP2358, 08 August 2013 - 08:12 PM.