Integrity of overhang after digestion - (Sep/07/2007 )
Hello, I am wondering how one can confirm that the overhang sequence of a restriction fragment after digestion with a type IIS restriction enzyme is correct? The type IIS Restriction enzymes cut at specific sites outside their recognition sequences. Can mass spec help?
you can ligate it to a compatible end and test for the completeness of the ligation. This is probably the easiest way. Mass spec would work, but seems like overkill, perhaps just because I don't have a mass spec in my lab.
Ligating will not provide direct evidence. In a recent ligation reaction, I noticed that the ligase catalyzed ligation between non-cohesive overhangs (with mismatched bases).
If Mass Spec can work, how can one go about it? I have no mass spec in my lab either.
Thanks for previous answer.
Are you sure? I’ve never seen that!
Sometimes it looks like fragments are ligated when run ligation reaction in a gel but they are not really ligated and if transform, you don’t obtain any colony.
If you add SDS and heat a little before running a gel, you eliminate that “background” in the gel.