A circular plasmid vector has been cut with a specific restriction enzyme to incorporate our gene of interest into it. How can we prevent that vector which is now linear from binding to itself and become circular again without enclosing our gene of interest after the addition of ligase enzyme?
Inhibition of religation is done with a phosphatase reaction step on the linearized vector. E.g with Calf intestine phosphatase (CIP) or antartic phospatase. The latter can be heat inactivated and doesnt require an extra purification.
Good luck
Always try a cloning strategy with 2 enzymes that generate non-compatible overhangs or the combination of an enzyme that generates an overhang with a blunt end generating enzyme. In the other case you have to treat your vector with a phosphatase, the most prominent is CIP (calf intestine phosphatase).
There are some special modifying enzymes which yield one end as flush and the other as sticky when you digest your vector. Hope that will help you out.
GOOD LUCK
Depends entirely on your cloning methodology. Unless you are using a blunt end cloning strategy or digestion with a single restriction enzyme, self-ligations should not be a problem. There are also excellent ligation-independent, recombination-based cloning strategies that eliminate the problems of recovering empty vector. GOOD LUCK
Hi chowdary,
Have you tried dephosphorylating your vector after digestion?
We do this routinely to prevent re-ligation of the vector and prior to ligation with the insert.
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