I have a doubt about a protocol I'm using for a ligation of a very large plasmid (15kbp circa) 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?


1 Answer 1


A plasmid vector will always be prone to recircularisation when a single restriction enzyme has been used, but recombinant plasmids will also be formed during the ligation. Alkaline phosphatase treatment can be used as you describe; you can also simply make sure that the insert fragment is present in high molar excess and be prepared to work harder at the end to find the recombinant plasmids. Neither approach is correct or incorrect, they both have their limitations.

Of course the best approach is to avoid this type of cloning altogether.

Added later

Incidentally, the wording of your question suggests that you think you may have more of a problem because the plasmid vector is a little larger than usual. Not so! Circularisation is favoured because the two ends are tethered so the effective concentration of each is high with respect to the other. As the plasmid gets bigger the tether is longer and the tethering effect will be less.


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