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.

    1. Standard Protocol for Restriction Enzyme Digests

      Let one of NEB's restriction enzyme experts help you improve your technique and avoid common mistakes in digest setup.

    2. Why is My Restriction Enzyme Not Cutting DNA

      Not getting the cleavage you expected? Let an NEB scientist help you troubleshoot your reaction.

    3. Restriction Enzyme Digest Problem: Too Many DNA Bands

      Are you finding unexpected bands in your digestion reaction? Here are some tips to help you determine the cause.

    4. What is Restriction Enzyme Star Activity?

      Learn what Star Activity is, why it is detrimental to accurate restriction enzyme digestion, and how NEB's HF™ enzymes are engineered to avoid it.

    5. Setting-up Restriction Enzyme Digests with RE-Mix® Master Mixes

      RE-Mix® Restriction Enzyme Master Mixes offer simplified reaction setup. Learn more about digesting DNA with RE-Mix.

    6. RE-Mix® Double Digest Protocol

      Double digests are now easier than ever! Learn how to set up your next double digest with RE-Mix®.

    7. Reduce Star Activity with High-Fidelity Restriction Enzymes

      NEB has engineered HF enzymes to eliminate star activity. Learn how, and what this means for your digests.

    8. NEB Restriction Enzyme Double Digest Protocol

      Double digestions can save you time, and this video can offer tips for how to achieve the best results, no matter which of NEB's restriction enzymes you're using.

    9. Restriction Enzyme Digest Protocol: Cutting Close to DNA End

      When cutting close to the end of a DNA molecule, make sure you know how many bases to add to the ends of your PCR primers.

    10. Restriction Enzyme Digestion Problem: DNA Smear on Agarose Gel

      Learn more about what causes this common problem, and how NEB's enzymes are QC'd to avoid DNA smearing.

    11. Cloning With Restriction Enzymes

      Restriction enzymes are an integral part of the cloning workflow, for generating compatible ends on fragments and vectors. This animation discusses three guidelines for determining which restriction enzymes to use in your cloning experiment.

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