Haelll Methyltransferase

Description

HaeIII Methyltransferase modifies the internal cytosine residue (C5) of the sequence GGCC.

Product Source

An E. coli strain that carries the cloned HaeIII modification gene from Haemophilus aegyptius (ATCC 11116).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
HaeIII 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 λ DNA in 1 hour at 37°C in a total reaction volume of 10 µl against cleavage by HaeIII restriction endonuclease.

Reaction Conditions

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

1X HaeIII Methyltransferase Reaction Buffer:
50 mM Tris-HCl
50 mM NaCl
10 mM DTT
pH 8.5 @ 25°C

HaeIII Methyltransferase is incubated with 1 µg of λ DNA in 10 µl of 1X HaeIII Methyltransferase Reaction Buffer, supplemented with 80 µM S-adenosylmethionine, for one hour at 37°C followed by 15 minutes at 65°C. The extent of protection is determined by the addition of 40 µl NEBuffer 2.1 and 10 units of HaeIII restriction endonuclease. Incubation for 1 hour at 37°C is followed by analysis on an agarose gel.

Storage Temperature

-20°C

Storage Conditions

50 mM Tris-HCl
1 mM DTT
10 mM EDTA
200 μg/ml BSA
50% Glycerol
50 mM KCl
pH 7.4 @ 25°C

Heat Inactivation

65°C for 20 min

Notes

  1. HaeIII Methyltransferase protects DNA against cleavage by NotI.

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. Does HaeIII Methyltransferase require DTT?
  4. Can HaeIII Methyltransferase be heat inactivated?
  5. Does HaeIII Methyltransferase require MgCl2?
  6. What is the molecular weight of HaeIII Methyltransferase?

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.

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.
  • 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.
  • Non-Specific RNase Activity (16 hour, RNA):
    The RNA is tested in a reaction under standard reaction conditions. After incubation for 16 hours there is no detectable degradation of the RNA 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.

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.

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.