Self-ligation from a Double Digest
Posted 10 May 2010 - 06:55 PM
I've known of self ligation from a single digestion and many avoid that by using two different REs.
I'm designing a gene to be synthesized and now realized I have inserted AvrII and NheI to the 5' and 3' of the DNA. These ends will be cut and inserted into a vector.
Will I be lucky as to get a successful ligation or will I almost always end up with self ligation similar to that of a single digest? Problem is I have sent it to be processed.
Posted 10 May 2010 - 11:15 PM
Posted 12 May 2010 - 01:24 AM
Don't worry, all you have to do is dephosphorylate your vector and if that goes well, you should not have any self-ligated vectors. The only thing that can happen then is that your insert can be ligated in your vector in the sense or antisense direction, so you'll have to check some clones to get one in the good direction.
Thank you dpo. Do I have to add PNK in the subsequent ligation reaction then?
Posted 12 May 2010 - 01:35 AM