question in ligation - (Sep/06/2008 )
Hi every one, i have some question that is:
I want to do ligation for my gene with pET28b(+) vector, can i use ligation protocol supplied with CloneJET Kit for pJET1.2 vector?
CloneJET Kit uses a (pre) blunt end pJET1.2 vector to ligate a blunt end inserts. While the pET28b(+) vector, is... well an uncut plasmid vector. Which you will need to digest first before use.
You could cut the pET28b vector and than blunt end it. This will allow you to use a blunt end ligation similar to that described in the protocol of CloneJET kit. However note pET28b does not have a lethal kill gene to remove any recircularised empty vectors. So a blunt end ligation with pET28b+ will be more difficult compared to a sticky end ligations.