Restriction to remove background - (Jul/18/2014 )

Hi, when you ligate an insert into a plasmid with two different enzymes and these enzymes are far apart, would it be sensible to do a restriction digest with a enzyme that creates blunt end cuts between the two sites used of restriction, and by this reasoning only the self-ligated empty plasmids (no desired insert) would be cut by the enzyme and therefore reducing the background. then i would heat inactivate the enzyme.

has anyone done this? would the ligation buffer interfere with the restriction?

thanks

This makes  perfect sense. You can do these cuts at the same time you are doing other restriction cuts. Blunt cuts, as you suggest, work better than overhang cuts, since they ligate poorly, especially without PEG in the ligation mix. I don't understand what your comment about the ligation buffer has to do with it.

@phage: I think Ray was meaning to do the digest after ligation and thereby not run the risk of the blunt ends re-ligating at all.

correct, although it would have to be a heat inactivable enzyme. or maybe not as long as it doesn't interfere with the transformation efficiency.

If you use normal ligase (not quick ligase with PEG) blunt ligation is so inefficient that it can be ignored. A 5-15 minute room temperature ligation suffices for most overhang DNA ligations, and it will not ligate blunt ends to any measurable extent.

If you add a blunt cutter to the ligation reaction, then the insert DNA must also not contain a cut site, a condition that is difficult to arrange ahead of time, and makes  you have to think about details of each ligation individually.