New England Biolabs UK Ltd CST - Cell Signaling Technology Biohelix Corp
Search:
Product CatalogueNew from New England Biolabs...Special OffersTechnical ResourcesContact UsMy Account
home > Product Catalogue > New England Biolabs > DNA Modifying Enzymes and Cloning > Methyltransferases > AluI Methyltransferase
AluI Methyltransferase
Cloned At NEBRecombinant SourceSAM37
Catalog # Size Concentration Price(£) Qty
M0220L 500 units 5,000 units/ml 200.00
M0220S 100 units 5,000 units/ml 50.00
Download:MSDS PDF


Methylation Site:



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

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

Reagents Supplied:
AluI Methyltransferase Reaction Buffer (10X)
S-adenosylmethionine (SAM)


Reaction & Storage Conditions


Reaction Conditions:
1X AluI Methyltransferase Reaction Buffer
Supplemented with 32 μg/ml S-adenosylmethionine (SAM)
Incubate at 37°C.

1X AluI Methyltransferase Reaction Buffer:
50 mM Tris-HCl
5 mM 2-Mercaptoethanol
10 mM EDTA
pH 7.5 @ 25°C

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.

Protection Assay Conditions: 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.

Concentration:
5,000 units/ml

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

Storage Temperature:
-20°C


Notes


General notes:
  1. Storage of SAM: S-adenosylmethionine is stored at �20°C as 32�mM solution dissolved in sulfuric acid (0.005�M) and 10% ethanol. SAM in this solution stored under ideal conditions remains active for up to 6�months. SAM is unstable at (pH 7.5), 37°C, and should be replenished for reactions incubated longer than 4 hours. Many problems in achieving complete digestion can be alleviated by using fresh SAM.
  2. AluI methylated DNA is mcrB sensitive.

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?

Quality Control for Current Lot


Quality control values for a specific lot can be found on the datacard which accompanies each product.

16-Hour Incubation::
Incubation of 40 units of AluI Methyltransferase with 1 μg λ DNA in 50 μl of 1X NEBuffer 2 at 37°C resulted in no detectable endonuclease contamination.

Exonuclease Activity::
Incubation of 40 units of AluI Methyltransferase with 1 μg sonicated 3H DNA (105 cpm/μg) for 4 hours at 37°C in 50 μl NEBuffer 2 released < 0.2% of the total radioactivity.


References


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


Reagents Sold Separately


S-adenosylmethionine (SAM)