Self-ligation from a Double Digest - (May/10/2010 )
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.
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.
dpo on May 11 2010, 03:15 PM said:
Thank you dpo. Do I have to add PNK in the subsequent ligation reaction then?
No, you don't have to add PNK. After digestion of your insert with AvrII and NheI, there will be phosphate groups on the 5' ends of this DNA. During your ligation, only one strand of your insert DNA will be ligated to your vector DNA at each side of the insert, but when you do the transformation, the bacteria will correct this and 'repare' the nick in the DNA. However, if you add PNK, you will phosphorylate your vector again and the result will be self-ligated plasmid.
Noted and understood. Thank you dpo.