Feb 16, 2022
Version 4
In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386) V.4
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
External link: https://www.neb.com/protocols/2014/05/01/in-vitro-digestion-of-dna-with-cas9-nuclease-s-pyogenes-m0386
Protocol Citation: New England Biolabs 2022. In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386). protocols.io https://dx.doi.org/10.17504/protocols.io.be6fjhbnVersion created by Breton Hornblower
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: April 16, 2020
Last Modified: February 16, 2022
Protocol Integer ID: 35751
Keywords: Cas9, S. Pyogenes, in vitro digestion, sgRNA,
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).
Guidelines
REFERENCES:
- Jinek et al. (2012) Science 337 (6096) 816-821.
- Larson et al. (2013) Nature Protocol 8 (2180-2196).
- Mali et al. (2013) Science 339 (6121): 823-826.
Materials
MATERIALS
Cas9 Nuclease, S. pyogenes - 70 pmolNew England BiolabsCatalog #M0386S
Proteinase K, Molecular Biology Grade - 2 mlNew England BiolabsCatalog #P8107S
HiScribe T7 Quick High Yield RNA Synthesis Kit - 50 rxnsNew England BiolabsCatalog #E2050S
Nuclease-free WaterNew England BiolabsCatalog #E7764
REQUIRED MATERIALS:
- NEBuffer 3.1
- 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
- E.g. Agilent Bioanlyzer or similar
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
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 1 micromolar (µM) in 1 X Buffer 3.1 and used immediately. If the 1 µM dilution will be stored at -20 °C , it should be diluted using Diluent B (NEB #B8002S): 300 millimolar (mM) NaCl , 10 millimolar (mM) Tris-HCl , 0.1 millimolar (mM) EDTA , 1 millimolar (mM) DTT , 500 μg/ml BSA and 50 % glycerol (7.4 @ 25 °C ) prior to the reaction assembly.
Prepare 300 nanomolar (nM) sgRNA by diluting the stock with nuclease-free water On ice .
Prepare 30 nanomolar (nM) substrate DNA with a single target sequence by diluting the stock with nuclease-free water On ice .
Assemble the reaction at Room 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 00:10:00 at 25 °C .
Add 3 µL 30 nM substrate DNA (3 nM final).
Mix thoroughly and pulse-spin in a microfuge.
Incubate at 37 °C for 00:15:00 .
Add 1 µL Proteinase K to each sample. Mix thoroughly and pulse-spin in a microfuge.
Incubate at Room temperature for 00:10:00 .
Proceed with fragment analysis.