Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Biology

Clarification on digesting a ligation mixture! - (Aug/01/2012 )

Hi all (and badcell!)

I would like to requote badcell's post!

badcell on Wed Jan 26 18:41:53 2005 said:

In theory it's not absolutely necessary to dephosphorylate if you are using two REs with cohesive ends, but in fact, if the restriction efficiency of the enzymes is not 100%, you will get some molecules which have been cut only with one or the other enzyme, so they will religate if they are phosphorylated. One useful trick when cloning using two different sites is checking if you loose any site in the vector multiple cloning site (if any site is between the two REs you are using and not anywhere else in the vector). If this is the case, and this site is not found neither in your insert, you can digest your ligation with that enzyme and you will cut only your religated plasmids and not the ones with your insert, which will not have that site. So you will get only colonies with the insert.

This actually sounds great and i want to try it! But i do have a question... please correct me if am wrong! When we RE double digest the vector, we always gel purify it. So i am guessing every unwanted bit will be gone (including the middle bit between the two RE's we used!). So if the vector gets religated (during the ligation!), then there wont be a middle portion!! Right?? Can anyone please explain this to me?

Thanks in advance!



In a double digest if one of the enzymes doesnt cut all of the sites, then your linearized DNA will run the same as the double digested on a gel and you will cut it out together with what you actually want. What badcell described will eliminate this problem however in most cases for most enzymes 15 minutes will digest your vector entirely.