Which enzyme for repairing nicks in ligated constructs? - (Nov/26/2007 )
I have adaptors ligated to a DNA fragment. But the adaptors are not 5' phosphorylated so there are still nicks in the ligated construct.
XXXXXX-OH .... P-DNA-OH .... OH-YYYYYYY
YYYYYYY-OH ....OH-DNA-P ...... OH-XXXXXX
I have come across several protocols for repairing the nicks.
1. DNA Pol I from E Coli.
2. DNA Pol I from E Coli + E coli ligase
3. Bst DNA Pol, large fragment.
What would your preference be here????
Phosphorylating the adaptors prior to ligation would make more sense. PNK will do this. Also, do you need to do this? E. coli will gladly repair nicks in circular DNA plasmids if the overlapping region is sufficiently long (probably 10-12 bp is enough). Of course you may not be making a plasmid.