Puzzling difficulty cutting out one gene sequence for another - (Aug/08/2013 )

Hey guys,

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?

Thanks.

It sounds like your pAB is not being cut properly.  How are you checking the digest is working properly.  Are you sure that your insert (C) is being digested correctly too?

I'm sure that pAB is since I can see the proper bands on a gel.  Since I am cutting C with the same enzymes, I have just assumed that it is being cut, though I have not been running it on a gel because I'm being forced to work with low quantities of DNA.

Also, if my pAB were not being cut, then why is my backbone-only control not producing colonies?

Ah, I was assuming your backbone control was cut vector without ligase.  The vector would need to be single cut for the re-ligation to occur.

I take it that you mean that you are gel purifying the backbone after digestion, when you say that you are cutting it out?

Do your inserts have several bases outside the cut sites to allow the REs to bind to the sequence?