Do I need CIP before my ligation - (Dec/29/2005 )
Hi Dear ALL,
I cut my vector with XhoI and XmnI, and cut the insert with XhoI and NruI, both XmnI and NruI will give a blunt end. After gel purification of them, can I ligate the insert and vector directly or should I do more work such as CIP for the vector or insert because of the blunt end?
thanks for any kind help!
what is CIP ?
I think you shouldn't need to CIP treat.
The ends are incompatable.
CIP or SIP are intestinal phosphatases used to remove the phosphate groups that hang off the end of dna strands. If you are doing a blunt end ligation you should definitely "CIP" the vector to stop vector religation. When you CIP the vector you will favor the ligation reaction that puts you phosphorylated insert into the unphosphorylated vector. You will have to sequence (or perform restriction digests on) the ligated vector to make sure your insert is in the desired orientation
As either ends of your vector are not same you need not CIP treat your vector.