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Restriction Endonucleases >
Restriction Endonucleases >
MmeI |
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Prices are in US dollars and valid only for US orders.
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
A E. coli strain that carries the MmeI gene from Methylophilus methylotrophus
Reagents Supplied:
NEBuffer 4
S-adenosylmethionine (SAM)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | Not Recommended | | NEBuffer 2: | | Not Recommended | | NEBuffer 3: | | Not Recommended | | NEBuffer 4: | | 100% |
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Methylation Sensitivity:
| dam methylation: Not sensitive | | dcm methylation: Not sensitive | | CpG methylation: Blocked by overlapping |
Heat Inactivation:
80°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 1.00 unit(s)
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 4
Supplemented with 50 μM S-adenosylmethionine (SAM)
Incubate at
37°C.
1X NEBuffer 4:
20 mM Tris-acetate
50 mM potassium acetate
10 mM Magnesium Acetate
1 mM Dithiothreitol
pH 7.9 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to digest 1 µg of ΦX174 RF I DNA in 1 hour at 37°C in 50 µl of reaction buffer.
Concentration:
2,000 units/ml
Unit Assay Substrate:
ΦX174 DNA
Storage Conditions:
10 mM Tris-HCl
300 mM NaCl
1 mM DTT
0.1 mM EDTA
500 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent B
Notes
Application notes:- Excess MmeI blocks cleavage.
- Reactions using MmeI should be done at or near stoichiometric concentrations.
- Complete cleavage occurs within 15 minutes at 37°C.
- Significant cleavage occurs on ice and at 50°C.
- MmeI activity is inhibited by high ionic strength (> 200 mM).
- SAM must be present in a reaction at a concentration of 50 µM or higher for optimal cleavage.
- Potassium is necessary for optimal cleavage efficiency.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Ligation and Re-cutting:
After a 1-fold overdigestion with MmeI, > 95%
of the DNA fragments can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 μM)
at 16ºC. Of these ligated fragments, < 5% can be recut with MmeI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 2 units of MmeI incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 100 units of MmeI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.1% of the total radioactivity.
Reagents Sold Separately
NEBuffer 4
S-adenosylmethionine (SAM)
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 7,115,407
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