Product Class: Methylase

dam Methyltransferase

recombinant unique buffer incubation temp sam
M0222damMethyl_thumb
Catalog #SizeConcentration
M0222S500 units8,000 units/ml
M0222L2,500 units8,000 units/ml

Description

dam Methyltransferase modifies the adenine residue (N6) of the sequence GATC.

Product Source

An E. coli strain that carries plasmid pTP166 carrying the dam modification gene of E. coli

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
dam Methyltransferase Reaction Buffer10X
S-adenosylmethionine (SAM)-2032 mM

Properties and Usage

Unit Definition

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

Reaction Conditions

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

1X dam Methyltransferase Reaction Buffer:
50 mM Tris-HCl
5 mM β-ME
10 mM EDTA
pH 7.5 @ 25°C

dam Methyltransferase is incubated with 1 µg of λ DNA in 10 µl of 1X dam Methyltransferase Buffer, supplemented with 80 µM S-adenosylmethionine, for 1 hour at 37°C followed by 15 minutes at 65°C. The extent of protection is determined by addition of 40 µl 1X NEBuffer 3 supplemented with 10 mM MgCl2 and 10 units of MboI restriction endonuclease. Incubation at 37°C for 1 hour is followed by analysis on an agarose gel.

Storage Conditions

50 mM Tris-HCl
50 mM KCl
1 mM DTT
10 mM EDTA
200 μg/ml BSA
50% Glycerol
pH 7.5 @ 25°C

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.
  • 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.
  • Functional Test (Methyltransferase):

    The Methyltransferase is tested in a reaction with Lambda DNA and SAM, followed by a second reaction with the appropriate restriction enzyme. After incubation the extent of methylation is determined by agarose gel 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.

Supporting Documents

Material Safety Datasheets

The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

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.
  1. Is S-adenosylmethionine (SAM) supplied with the Methyltransferase?
  2. What should be considered if the methylation is not going to completion?
  3. Can dam Methyltransferase be heat inactivated?
  4. Does dam Methyltransferase require MgCl2?
  5. Is dam Methyltransferase sensitive to salt?
  6. Will DpnI cleave dam methylated DNA?

Usage Guidelines & Tips

Always add the SAM to the reaction buffer just before doing the methylation reaction, because the SAM is unstable in an aqueous solution.

Keep the SAM frozen at -20C for a longer shelf life, fresh SAM is important for optimal methylation reactions.