Large plasmid ligation: do i need Alkaline phosphatase?

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Large plasmid ligation: do i need Alkaline phosphatase?

Postby Leopold_Mexico » Jul 12 2017 11:31 am

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 :roll: . Am I mistaken or the protocol is incorrect?

Cheers! :wink:
Last edited by Leopold_Mexico on Jul 15 2017 1:49 am, edited 1 time in total.
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Re: Large plasmid ligation: do i need Alkaline phosphatase?

Postby relaxin » Jul 13 2017 11:32 am

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.
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