In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)
New England Biolabs
Abstract
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).
Before 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.
- If planning to use higher concentration Cas9 Nuclease, S. pyogenes (NEB #M0386T and NEB #M0386M) for in vitro digestion of DNA, the enzyme can be diluted to
1micromolar (µM)in1Xand used immediately. If the 1 µM dilution will be stored at-20°C, it should be diluted using Diluent B (NEB #B8002S):300millimolar (mM),10millimolar (mM),0.1millimolar (mM),1millimolar (mM),500μg/mland50%(7.4@25°C) prior to the reaction assembly.
Steps
Prepare 300nanomolar (nM) by diluting the stock with nuclease-free water 37On ice.
Prepare 30nanomolar (nM) with a single target sequence by diluting the stock with nuclease-free water 37On ice.
Assemble the reaction at 37Room temperature in the following order:
| A | B |
|---|---|
| COMPONENT | VOLUME (for 30 µl reaction) |
| Nuclease-free water | 20 µl |
| NEBuffer 3.1 | 3 µl |
| 300 nM sgRNA | 3 µl (30 nM final) |
| 1 µM Cas9 Nuclease, S.pyogenes (M0386S) | 1 µl (~30 nM final) |
| Reaction volume | 27 µl |
*The sgRNA and nuclease-free water are not included.
Pre-incubate for 0h 10m 0s at 25°C.
Add 3µL (3 nM final).
Mix thoroughly and pulse-spin in a microfuge.
Incubate at 37°C for 0h 15m 0s.
Add 1µL to each sample. Mix thoroughly and pulse-spin in a microfuge.
Incubate at 37Room temperature for 0h 10m 0s.
Proceed with fragment analysis.
