EcoGII Methyltransferase

Description

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

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 CutSmart® 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), ggenomic DNA (Human Embryonic Kidney (HEK293) cells, Mouse Ear and Tail, Rat Liver and Kidney, N6-methyladenine-free λ DNA #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:

Store at (°C)Concentration
CutSmart® Buffer-2010X
S-adenosylmethionine (SAM)-2032 mM

Properties and 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 CutSmart® Buffer
Supplement with 32 mM S-adenosylmethionine (SAM)
Incubate at 37°C

1X CutSmart® Buffer:
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 μg/ml BSA
pH 7.9 @ 25°C

Storage Temperature

-20°C

Storage Conditions

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 min

Unit Assay Conditions

EcoGII Methyltransferase is incubated with 100 ng of a FAM-labeled dsDNA substrate (80 bp) in 20 µl of 1X CutSmart 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.

Notes

  1. 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. 
  2. For use of methylation reaction the SAM should be diluted 1:200 in H2O to a final concentration of 160 µM.
  3. 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.

Interactive Tools

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Endonuclease Activity (Nicking):
    The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
  • Exonuclease Activity (Radioactivity Release):
    The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
  • Functional Test (Methyltransferase):
    The Methyltransferase is tested in a reaction with a FAM-labeled dsDNA oligo and SAM, followed by a second reaction with the appropriate restriction enzyme. After incubation, the extent of methylation is determined by capillary electrophoresis.
  • Non-Specific DNase Activity (16 hour):
    The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
  • Protein Purity (SDS-PAGE):
    The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
  • RNase Activity (16 Hour Digestion):

    The product is tested in a reaction containing a RNA substrate.  After incubation for 16 hours there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

Certificate of Analysis

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

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]

Safety Data Sheet

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