subcloning into a single NotI site - (Jun/08/2005 )
I am in the same situation right now...been banging my head....just want to find out if that NotI cloning worked out on your side. Let me know. As for me...I m gonna push the ratio and try again...1:10 vector:insert and I may even try cloning without CIP cause I heard CIP treatment can sometimes work against the cloning....
If you digest your vector only with NotI, you certainly have to CIP. Otherwise you will only find self-ligated vector, since this reaction is far more efficient then integrating the insert.