Product Class: Other

EcoGII Methyltransferase
neb31 cloned at NEB recombinant 37 65 Heat SAM

Available in higher concentration. Please inquire.

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.


M0603 cutsite image

Product Introduction

EFI_EcoGII_Methyltransferase

EcoGII Methyltransferase is a non-specific methyltransferase that modifies adenine residues (N6) in any sequence context.

Catalog # Size Concentration
M0603S 200.0 units 5000 units/ml

Product Information

Description

EcoGII Methylation of Various Substrates

Substrate % Methylation*
DNA Substrates1
Plasmid DNA >75%
Genomic DNA >50%
ds DNA oligo >50%
ss DNA oligo >50%
DNA/RNA hybrid >50%
RNA Substrates2
Total RNA 5-10%
mRNA (in vitro transcribed) 5-10%
DNA/RNA hybrid 5-10%

*Methylation Conditions: Methylation of 1 µg substrate with 5 U EcoGII Methyltransferase in 1X rCutSmart™ Buffer supplemented with 160 µM SAM for 60 minutes at 37°C. Following methylation, samples were column purified, digested to nucleosides and analyzed by LCMS to determine the ratio of N6mA to A (% methylation).

1 Tested substrates include: plasmid DNA (pBR322 #N3033, pUC19 #N3041), genomic DNA (Human Embryonic Kidney (HEK293) cells, Mouse Ear and Tail, Rat Liver and Kidney, λ DNA (dam-) #N3013), ds DNA oligo (80 nucleotides), ss DNA oligo (60 nucleotides), DNA/RNA hybrid (80 nucleotides), total RNA (HeLa cell RNA, Mouse Kidney RNA, Rat Brain RNA), mRNA (in vitro transcribed 1.8 kb FLuc mRNA).

2 Methylation levels of >20% have been observed by using less substrate (250 ng) and increasing the amount of enzyme (15 U).

Methylation levels can be increased by decreasing the amount of substrate, increasing the amount of enzyme and/or increasing the length of the methylation reaction.

Product Source

An E. coli strain that carries the cloned EcoGII methyltransferase gene from E. coli (strain C227-11)(1).
This product is related to the following categories:
Methyltransferases for Epigenetics,
Methyltransferases Products,

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 protect 100 ng FAM-labeled dsDNA in 30 minutes at 37°C in a total reaction volume of 20 µl against cleavage by MboI restriction endonuclease.

Reaction Conditions

1X rCutSmart™ Buffer
Supplement with 160 µM S-adenosylmethionine (SAM)
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)

Storage Buffer

10 mM Tris-HCl
250 mM NaCl
1 mM DTT
0.1 mM EDTA
0.15% Triton® X-100
180 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

65°C for 10 minutes

Unit Assay Conditions

EcoGII Methyltransferase is incubated with 100 ng of a FAM-labeled dsDNA substrate (80 bp) in 20 µl of 1X rCutSmart Buffer supplemented with 160 µM SAM, for 30 minutes at 37°C followed by 10 minutes at 65°C. The extent of protection is determined by addition of 5 units of MboI restriction endonuclease (incubation at 37°C for 30 minutes, 65°C for 20 minutes) followed by capillary electrophoresis analysis to resolve the reaction products, assign the cleaved and uncleaved species and quantitate their abundance.

Product Notes

  1. For use of methylation reaction the SAM should be diluted 1:200 in H2O to a final concentration of 160 µM.
  2. EcoGII Methyltransferase is sensitive to salt. Make sure the DNA solution is low in salt concentration or that it makes up only a small percentage of the final reaction volume. If salt is a problem, reduce the salt concentration by drop dialysis. 

References

  1. Fang, et al (2012). Nature Biotechnology. 30, 1232-1239.
  2. Hoffman, J.L. (1986). Biochemistry. 25, 4444-4449.

Protocols, Manuals & Usage

Protocols

  1. Recommended Protocol for 3H labeling of DNA
  2. Recommended Protocol for Methylation of Genomic DNA

FAQs & Troubleshooting

FAQs

  1. Will any NEB methyltransferases (methylases) work on RNA?
  2. What is the activity of EcoGII Methyltransferase at temperatures other than 37°C?
  3. Is S-adenosylmethionine (SAM) supplied with the Methyltransferase?
  4. Does EcoGII Methyltransferase require BSA?
  5. Does EcoGII Methyltransferase require MgCl2?
  6. What is the activity of EcoGII Methyltransferase in other buffers?
  7. What is the molecular weight of EcoGII Methyltransferase?
  8. Can EcoGII Methyltransferase be heat inactivated?
  9. Is EcoGII Methyltransferase sensitive to salt?
  10. Will EcoGII Methyltransferase methylate ssDNA?
  11. Will EcoGII Methyltransferase methylate DNA/RNA hybrids?
  12. Will EcoGII Methyltransferase methylate gDNA?
  13. Will EcoGII Methyltransferase methylate RNA?
  14. Will EcoGII methylation block restriction enzymes? 
  15. How can I increase methylation of my substrate by EcoGII Methyltransferase?
  16. Can I purchase large amounts of an existing Enzyme for Innovation?
  17. What are Enzymes for Innovation?
  18. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?

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.

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