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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?
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.
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.
Cloning, Expression and Characterization of a Lipase Encoding Gene from Human Oral Metagenome
The human oral metagenomic DNA cloned into plasmid pUC19 was used to construct a DNA library in Escherichia coli. Functional screening of 40,000 metagenomic clones led to identification of a clone LIP2 that exhibited halo on tributyrin agar plate. Sequence analysis of LIP2 insert DNA revealed a 939 bp ORF (omlip1) which showed homology to lipase 1 of Acinetobacter junii SH205. The omlip1 ORF was cloned and expressed in E. coli BL21 (DE3) using pET expression system. The recombinant enzyme was purified to homogeneity and the biochemical properties were studied. The purified OMLip1 hydrolyzed p-nitrophenyl esters and triacylglycerol esters of medium and long chain fatty acids, indicating the enzyme is a true lipase. The purified protein exhibited a pH and temperature optima of 7 and 37 °C respectively. The lipase was found to be stable at pH range of 6–7 and at temperatures lower than 40 °C. Importantly, the enzyme activity was unaltered, by the presence or absence of many divalent cations. The metal ion insensitivity of OMLip1offers its potential use in industrial processes.
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