ligating blunt ends of a linearized plasmid, is kinase necessary? - (Mar/04/2013 )
A quick cloning question:
I want to delete part of a plasmid. I double digest with to REs which result in sticky ends. I gel-purify the desired linearized fragment. I blunt-end with T4 polymerase and the ligate the ends together by T4 ligase.
Before the ligation, do I need to use T4 kinase to add phosphate groups to 5' ends?
No, T4 ligase is sufficient for blunt end ligation. The provided ATP will provide the phosphate linkage.