# In vitro digestion of DNA with sgRNA/Cas9

[EcoEvoDevo Lab](https://hackmd.io/@EcoEvoDevoLab/AngeliniLab)

Updated 27 June 2022

Based on the protocol from [New England Biolabs](https://www.protocols.io/view/in-vitro-digestion-of-dna-with-cas9-nuclease-s-pyo-b6qpvrmzgmkn/v4) for their Cas9 reagent (catalog number M0386). Exercise RNase precautions.


https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626505/

:::info
Use a 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](http://nebiocalculator.neb.com/).
:::

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

| reagent | volume (for a 30 µl reaction) |
| --------------------------------------- | --------------------------- |
| nuclease-free water | 13 µl |
| NEBuffer 3.1 | 3 µl |
| 1 µg/µl sgRNA | 1 µl |
| 200 ng/µl Cas9 | 3 µl (20 ng/ul final) |
| PCR product | 10 µl |

6. Mix thoroughly and pulse-spin in a microfuge.
7. Incubate at 37 °C for 2 hours.
8. Incubate at 95-98 °C for 10 min.
9. Verify the results of the digestion by [agarose gel electrophoresis](https://hackmd.io/@EcoEvoDevoLab/agarosegels).

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