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DNA Modifying Enzymes and Cloning >
Methyltransferases >
BamHI Methyltransferase |
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Prices are in US dollars and valid only for US orders.
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Methylation Site:
Description:
BamHI Methyltransferase modifies the internal cytosine residue (N4) of the sequence GGATCC.
Source:
A E. coli strain that carries the cloned BamHI modification gene from Bacillus amyloliquefaciens H (ATCC 49763)
Reagents Supplied:
BamHI Methyltransferase Reaction Buffer (10X)
S-adenosylmethionine (SAM)
Reaction & Storage Conditions
Reaction Conditions:
1X BamHI Methyltransferase Reaction Buffer
Supplemented with 32 mM S-adenosylmethionine (SAM)
Incubate at
37°C.
1X BamHI Methyltransferase Reaction Buffer:
50 mM Tris-HCl
5 mM Dithiothreitol
10 mM EDTA
pH 7.5 @ 25°C
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 BamHI restriction endonuclease.
Protection Assay Conditions: BamHI Methyltransferase is incubated with 1 µg λ DNA in 10 µl 1X BamHI 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 BamHI Methyltransferase is determined by the addition of 40 µl NEBuffer 1 supplemented with 10 mM MgCl2 and 10 units of BamHI restriction endonuclease. Incubation at 37°C for 30 minutes is followed by analysis on agarose gels.
Concentration:
4,000 units/ml
Storage Conditions:
50 mM Tris-HCl
1 mM Dithiothreitol
10 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- 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.
FAQs
- Is S-adenosylmethionine (SAM) supplied with the Methyltransferase?
- What should be considered if the methylation is not going to completion?
- What is the molecular weight of BamHI Methyltransferase?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Exonuclease Activity:
Incubation of a 50 μl NEBuffer 2 [50 mM NaCl, 10 mM Tris-HCl (pH 7.9 @ 25°C), 10 mM MgCl2, 1 mM DTT] reaction containing 30 units of BamHI Methyltransferase with 1 μg of a mixture of single and double-stranded [3H] E. coli DNA (105 cpm/μg) for 4 hours at 37°C released < 0.3% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 µl NEBuffer 2 reaction containing 15 units of BamHI Methyltransferase with 1 μg of λ DNA (HindIII digest) for 16 hours at 37°C resulted in no degradation of the DNA as determined by visualization of clear and sharp bands following gel electrophoresis.
References
- Hoffman, J.L. (1986) Biochemistry, 25, 4444-4449.
Reagents Sold Separately
BamHI Methyltransferase Reaction Buffer
S-adenosylmethionine (SAM)
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