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  • In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)

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    Cas9 Nuclease, S. pyogenes, (Cas9) is a double-stranded DNA endonuclease that is guided to its target by sequence complementarity of a small RNA loaded into the protein. This protocol describes how to digest double-stranded DNA in vitro using Cas9 and a single guide RNA (sgRNA).

    Required Materials:

    • Cas9 Nuclease, S. pyogenes (NEB #M0386 )
    • 10X Cas9 Nuclease Reaction Buffer
    • Nuclease-free water
    • sgRNA containing the targeting sequence in the region of interest
      • sgRNAs can be generated by in vitro transcription using the HiScribe T7 Quick High-Yield RNA synthesis Kit (NEB #E2050) using linearized plasmid, PCR products, or oligonucleotides as templates
      • sgRNAs must contain sequence complementary to the target DNA (1,2)
      • For information on design of sgRNA transcription templates please visit Addgene
    • DNA substrate containing the target sequence
    • The substrate DNA can be circular or linearized plasmid, PCR products, or synthesized oligonucleotides

    Optional Materials:

    • Apparatus and reagents for DNA fragment analysis
      • E.g. Agarose gel electrophoresis apparatus
        • DNA Loading Dye (e.g. Gel Loading Dye, Purple (6X) (NEB #B7024S)
      • E.g. Agilent Bioanlyzer or similar

    Before You Start:

    • We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Further recommendations for avoiding ribonuclease contamination can be found here.
    • Reactions are typically 30 μl but can be scaled up as needed. Reactions should be assembled in nuclease-free microfuge tubes or PCR strip tubes.
    • It is essential to keep the molar ratio of Cas9 and sgRNA per target site at 10:10:1 or higher to obtain the best cleavage efficiency. A calculator can be found here.
    • Prepare 300 nM sgRNA by diluting the stock with nuclease-free water on ice.
    • Prepare 30 nM substrate DNA with a single target sequence by diluting the stock with nuclease-free water on ice.


    1. Assemble the reaction at room temperature in the following order:

    2. Component 30 µl reaction
      Nuclease-free water 20 µl
      10X Cas9 Nuclease Reaction Buffer 3 µl
      300nM sgRNA 3 µl (30 nM final)
      1 µM Cas9 Nuclease, S. pyogenes (M0386S) 1 µl (~30 nM final)
      Reaction volume 27 µl
      Pre-incubate for 10 minutes at 25⁰C
      30nM substrate DNA 3 µl (3 nM final)
      Total reaction volume 30 µl
      *The substrate DNA and sgRNA, and nuclease-free water are not included.

    3. Mix thoroughly and pulse-spin in a microfuge.
    4. Incubate at 37°C for 1 hour.
    5. Proceed with fragment analysis.


    1. Jinek et al. (2012) Science 337 (6096) 816-821.
    2. Larson et al. (2013) Nature Protocol 8 (2180-2196).
    3. Mali et al. (2013) Science 339 (6121): 823-826.