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  • Restriction Enzyme Digestion

    Restriction enzymes, first described in 1971, are bacterially derived enzymes that cleave DNA. Evolutionarily, restriction enzymes arose as a bacterial self-defense mechanism; the genomes of invading organisms would be degraded, leading to an inability to replicate. Type II restriction enzymes generally recognize an inverted repeat palindrome. This recognition site structure leads to a symmetrical cleavage of both DNA strands and results in either blunt- or sticky-ends of the digested DNA. Blunt ends are universally compatible with other blunt-ended DNA and possess a 5’ phosphate group to promote ligation. Sticky ends, on the other hand, are stretches of single-stranded DNA that is capable of self-ligation or ligation with a complementary region of DNA from another molecule or organism.

    1. Type II Restriction Enzymes

      Type II restriction enzymes are most commonly used for molecular biology applications, as they recognize stereotypical sequences and produce a predictable cleavage pattern. Learn more about how Type II REs work.

    2. Type I Restriction Enzymes

      Type I restriction enzymes are a group of endonucleases that recognize a bipartite sequence, but do not produce a predictable cleavage pattern. Learn more about how Type I REs work.

    3. Type III Restriction Enzymes

      Type III restriction enzymes are a group of endonucleases that recognize a non-pallindromic sequence, comprising two inversely oriented sites. Learn more about these poorly understood enzymes.

    4. 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.

    5. Why is My Restriction Enzyme Not Cutting DNA

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

    6. 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.

    7. 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.

    8. 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.

    9. RE-Mix® Double Digest Protocol

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

    10. 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.

    11. 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.

    12. 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.

    13. 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.

    14. 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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    Protocols for Restriction Enzyme Digestion