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EcoGII Methyltransferase

Catalog # Concentration Size List Price Quantity Your Price
M0603S 5000 units/ml 200 units $122.00
$109.80
Catalog # Size List Price Your Price
M0603S 200 units $122.00
$109.80
Catalog #
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*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

EFI_EcoGII_Methyltransferase

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

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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).
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  EcoGII Methyltransferase M0603SVIAL -20 1 x 0.04 ml 5,000 units/ml
  rCutSmart™ Buffer B6004SVIAL -20 1 x 1.25 ml 10 X
  S-adenosylmethionine (SAM) B9003SVIAL -20 1 x 0.1 ml 32 mM

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.

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Notes
  • Storage of SAM: S-adenosylmethionine (Sigma Catalog #A7007) is stored at –20°C as a 32 mM solution dissolved in 0.005 M sulfuric acid and 10% ethanol. Under these conditions SAM is stable for up to 6 months. SAM is unstable at 37°C (pH 7.5)(2), and should be replenished in reactions incubated longer than 4 hours. 
  • For use of methylation reaction the SAM should be diluted 1:200 in H2O to a final concentration of 160 µM.
  • 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
  • Fang, et al (2012). Nature Biotechnology. 30, 1232-1239.
  • Hoffman, J.L. (1986). Biochemistry. 25, 4444-4449.
Citations
Additional Citations
  • Stergachis AB, Debo BM, Haugen E, Churchman LS, Stamatoyannopoulos JA. (2020) Single-molecule regulatory architectures captured by chromatin fiber sequencing Science 368 (6498), 1449-1454.PubMedID: 32587015, DOI: 10.1126/science.aaz1646
  • Shipony Z, Marinov GK, Swaffer MP, et al. (2020) Long-range single-molecule mapping of chromatin accessibility in eukaryotes. Nat�Methods 17(3):319-327, DOI: 10.1038/s41592-019-0730-2
Quality Control Assay
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 Data Sheets
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