Product Class: Restriction Endonuclease

MspJI
neb31 cloned at NEB recombinant dil_B 37 65 Heat Epi

We are excited to announce that all reaction buffers are now BSA-free. NEB began switching our BSA-containing reaction buffers in April 2021 to buffers containing Recombinant Albumin (rAlbumin) for restriction enzymes and some DNA modifying enzymes. Find more details at www.neb.com/BSA-free.

NEB restriction endonuclease that recognizes the sequence CNNRNNNNNNNNN^NNNN_
Isoschizomers | Single Letter Code

Product Introduction

  • 100% activity in rCutSmart™ Buffer (over 210 enzymes are available in the same buffer) allowing for easier double digests
  • Specificity to epigenetically-relevant DNA modifications (5-mC and 5-hmC)
  • Restriction Enzyme Cut Site: CNNR(9/13)
Catalog # Size Concentration
R0661S 200.0 units 5000 units/ml
R0661L 1000.0 units 5000 units/ml

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Product Information

Description

MspJI, an EpiMark®, validated product is a modification-dependent endonuclease that recognizes mCNNR sites and generates a double-stranded DNA break on the 3´ side of the modified cytosine at N9/N13. The recognized cytosine modifications include C5-methylation (5-mC) and C5-hydroxymethylation (5-hmC) (1).

This enzyme is provided with an Enzyme Activator Solution which may be used for efficient digestion by MspJI.

The most common epigenetic modifications found in eukaryotic organisms are methylation marks at CpG or CHG sites. A subset of these modified sites are recognized and cleaved by MspJI.

At fully methylated CpG sites:
5´ . . . Y N mC G N R . . . 3´
3´ . . . R N G mC N Y . . . 5´

or CHG sites:
5´ . . . Y mC H G R . . . 3´
3´ . . . R G D mC Y . . . 5´

R = A or G
Y = C or T
H = A or C or T (not G)
D = A or G or T (not C) 

MspJI recognizes each hemi-methylated site individually and cleaves bidirectionally to generate 32-base or 31-base fragments, respectively. These fragments contain the central methylated site and have 4-base 5´ overhangs at each end. MspJI does not cleave unmodified DNA.

Product Source

An E. coli strain that carries the synthetic MspJI gene from Mycobacterium species JLS.
This product is related to the following categories:
Methylation Sensitive and Methylation Dependent Restriction Enzymes for Epigenetics,
Restriction Enzymes for Epigenetic Analysis,
Restriction Endonucleases H M Products,
DNA Methylation Analysis,
This product can be used in the following applications:
Restriction Enzyme Digestion

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Unit Definition

One unit is defined as the amount of enzyme required to digest 1 µg of pBR322 (dcm+) DNA in 1 hour at 37°C in a total reaction volume of 50 µl.

Reaction Conditions

1X rCutSmart™ Buffer
Supplement with 1X Enzyme Activator Solution
Incubate at 37°C

1X rCutSmart™ Buffer
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 µg/ml Recombinant Albumin
(pH 7.9 @ 25°C)

Activity in NEBuffers

NEBuffer™ r1.1: <10%
NEBuffer™ r2.1: <10%
NEBuffer™ r3.1: <10%
rCutSmart™ Buffer: 100%

Diluent Compatibility

Storage Buffer

10 mM Tris-HCl
300 mM NaCl
1 mM DTT
0.1 mM EDTA
500 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

65°C for 20 minutes

Methylation Sensitivity

dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Not Sensitive

Product Notes

  1. This enzyme has been used for epigenetic analysis in which target DNA containing CpG sites methylated in both strands is cleaved by MspJI to generate 32 bp fragments containing the methylated CpG sites. The isolated 32 bp fragments can be sequenced to reveal 5-mC modification sites (Cohen-Karni et al., (2011) PNAS 108: 11040-11045).
  2. Use of excess enzyme inhibits cleavage. Optimization of the amount of enzyme needed for complete digestion may be required for each substrate DNA.
  3. Star activity, including digestion of non-methylated DNA substrates, may result from non-optimal reaction conditions such as extended digestion time, high enzyme or activator concentration or a glycerol concentration of > 5%. Optimization of the amount of enzyme needed to achieve efficient specific cutting, while avoiding star activity, may be required for each substrate DNA.
  4. Optimal specificity of cleavage at 5-mC sites can be achieved by keeping the molar ratio of MspJI to 5-mC target sites between 0.1:1 to 1:1 in the presence of 1X Enzyme Activator Solution and digesting for up to 60 minutes. (The molarity of MspJI is ~7.4 μM).
  5. Not sensitive to CpG, dcm, or dam methylation.
  6. star activity may result from extended digestion, high enzyme concentration or a glycerol concentration of >5%

References

  1. Zheng, Y. et al. (2010). Nucl. Acids Res. doi:10, 1093/nar/gkq327.
  2. U.S. Publication No. 2010-0167942 Unpublished observation
  3. Cohen-Karni D et al. (2011). The MspJI family of modification dependent restriction endonucleases for epigenetic studies.. Proc. Natl. Acad. Sci. U.S.A.. Jul 5;108(27):, 11040-5.

Protocols, Manuals & Usage

Protocols

  1. Restriction Digest Protocol
  2. Protocol for generating 32 bp fragments from modified CpG sites in genomic DNA using MspJI, FspEI or LpnPI

Usage & Guidelines

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. How has MspJI been used for epigenetic studies?
  2. How can I minimize off target or star activity?
  3. Should I add more MspJI to get more cutting?
  4. Is this enzyme sensitive to dam, dcm or mammalian CpG methylation?
  5. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?
  6. Can Gel Loading Dye, Purple 6X (B7024) be stored in cold temperatures?

Troubleshooting

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Licenses

The purchase of this product conveys to the user the non-transferable right to use the purchased amount of product for Research Use Only. Commercial use of this product may require a license from New England Biolabs. Please contact busdev@neb.com for further details.

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