Large plasmid ligation: do i need Alkaline phosphatase?

Go to the profile of leopold_mexico
Jul 12, 2017

Hi everyone, i'm a new user.
I have a doubt about a protocol I'm using for a ligation of a very large plasmid (14kbp) with an insert: after digestion with a single-cut enzyme (sticky ends), the protocol passes directly to the purification of the plasmid without making any alkaline phosphatase. My doubt is during the ligation, the plasmid will self-ligated without accepting the insert . Am I mistaken or the protocol is incorrect?



Go to the profile of relaxin
relaxin 9 months ago

The protocol is incorrect. The vector should be dephosphorylated with alkaline phosphatase when it is cut with single enzyme. Phosphatase treatment is recommended even when the vector is cut with two different restriction enzymes, since there is always small amount of single-cut vector present.

If your vector is not in limited supply, I would recommend gel purification after dephosphorylation to remove intact vector.