Restriction Enzyme Digestion

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  • Restriction digestion of recombinant plasmid constructs provides a fast, cost-efficient method of gaining indirect sequence information. Multiple plasmid constructs can be analyzed simultaneously for the presence or absence of an insert, orientation of the insert, plasmid size, and some site-specific sequence data. Purified plasmid DNA is digested with 1 or more restriction enzymes (REs) selected to give a distinct DNA band pattern that is easily resolved by electrophoresis. Restriction mapping tools, such as NEBcutter®, allow the user to upload the expected sequence of a recombinant plasmid (vector + insert) and provide a predicted digestion pattern for all commercially available REs. Restriction enzymes that cut within the multiple cloning site (MCS) and result in a diagnostic pattern of 2-5 easy to resolve bands are typically selected. Methylation sensitivity of selected enzyme(s) should also be considered to assure that methylation from the host organism, such as dam, dcm, or CpG methylation, does not block cleavage. Comparison of analytical and predicted DNA digestion patterns indicates presence/absence of restriction sites at the expected position within the plasmid construct providing some information about the insert sequence. In addition, plasmid size can be confirmed by simply adding the size of each DNA band in the digestion. Finally, restriction digestion patterns can provide information regarding the diversity of a complex DNA library.

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    Protocols for Restriction Enzyme Digestion