AluI Methyltransferase

Description

AluI Methyltransferase modifies the cytosine residue (C5) of the sequence AGCT.

Product Source

An E. coli strain that carries the cloned AluI modification gene from Arthrobacter luteus (ATCC 21606).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
AluI 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 AluI restriction endonuclease.

Reaction Conditions

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

AluI Methyltransferase is incubated with 1 µg of λ DNA in 10 µl 1X AluI 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 AluI Methyltransferase is determined by  the addition of 40 µl NEBuffer 1 supplemented with 10 mM MgCl2 and 10 units of AluI restriction endonuclease. Incubation at 37°C for 30 minutes is followed by analysis on an agarose gel.

Storage Temperature

-20°C

Storage Conditions

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

References

  1. Hoffman, J.L. (1986). Biochemistry. 25, 444-4449.

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 AluI Methyltransferase be heat inactivated?
  4. Does AluI Methyltransferase require MgCl2?
  5. What is the molecular weight of AluI Methyltransferase?
  6. Can AluI methylated DNA be used to transform E. coli?
  7. Does methylation with AluI Methyltransferase block SacI restriction endonuclease?

Tech 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.

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.

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.