For two months now I have been trying to clone a 2 kb blunt fragment (NruI/HindIII, filled in with klenow) into a PvuII site of a 6.5 kb vector.
I am aware of the fact that blunt ligation are not as efficient as sticky end ligations, but still it should work.
There are some specific questions I would like to put forward:
1. Self ligation of the PvuII cut vector does not work. Is anyone aware of problems specific for a blunt PvuII site?
2. From the catalog a learned that blunt NruI sites are difficult to ligate. Does anybody know why, and if there is a way to imporve this?
Any further suggestions on how to improve blunt ligation efficiency are most welcom!
Dept. human Genetics
Leiden , The netherlands