EcoRI Methyltransferase

Description

EcoRI Methyltransferase modifies the internal adenine residue (N6) of the sequence GAATTC.

Product Source

An E. coli. strain that carries the cloned EcoRI modification gene from Escherichia coli RY13 (R. N. Yoshimori).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
EcoRI Methyltransferase Reaction Buffer-2010X
S-adenosylmethionine (SAM)-2032 mM

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme required to protect 1 μg λ DNA in 1 hour at 37°C in a total reaction volume of 10 μl against cleavage by EcoRI restriction endonuclease

Reaction Conditions

1X EcoRI Methyltransferase Reaction Buffer
Supplement with 80 μM S-adenosylmethionine (SAM)
Incubate at 37°C

1X EcoRI Methyltransferase Reaction Buffer:
50 mM Tris-HCl
50 mM NaCl
10 mM EDTA
pH 8 @ 25°C

EcoRI Methyltransferase is incubated with 1 µg of λ DNA in 10 µl 1X EcoRI Methyltransferase Buffer, supplemented with 80 µM S-adenosylmethionine, for one hour at 37°C followed by 15 minutes at 65°C. The extent of protection by EcoRI Methyltransferase is determined by the addition of 40 µl NEBuffer 2 supplemented with 10 mM MgCl2 and 5 units of EcoRI restriction endonuclease. Incubation at 37°C for 30 minutes is followed by analysis on an agarose gel.

Storage Temperature

-20°C

Storage Conditions

100 mM KPO4
200 mM NaCl
10 mM β-ME
0.1 mM EDTA
200 μg/ml BSA
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

65°C for 20 min

Molecular Weight

Theoretical: 37911 daltons

References

  1. Hoffman, J.L. (1986). biochemistry. 25, 4444-4449.

FAQs

  1. Is S-adenosylmethionine (SAM) supplied with the Methyltransferase?
  2. What should be considered if the methylation is not going to completion?
  3. Can EcoRI Methyltransferase be heat inactivated?
  4. Does EcoRI Methyltransferase require MgCl2?
  5. Will EcoRI methylation block the ApoI restriction enzyme?
  6. Will any NEB methyltransferases (methylases) work on RNA?

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Usage Guidelines & Tips

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.
  • 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.
  • 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.

Safety Data Sheet

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

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.