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Restriction Endonucleases >
Restriction Endonucleases >
MspI |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
A E. coli strain that carries the MspI gene from Moraxella species (ATCC 49670).
Reagents Supplied:
NEBuffer 4 (10X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 75% | | NEBuffer 2: | | 100% | | NEBuffer 3: | | 50% | | 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: Not sensitive |
Heat Inactivation:
80°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.50 unit(s)
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 4
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 λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl.
Concentration:
20,000 units/ml and 100,000 units/ml
Unit Assay Substrate:
λ DNA
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent A
Notes
General notes:- MspI is an isoschizomer of HpaII. When the external C in the sequence CCGG is methylated, MspI and HpaII cannot cleave. However, unlike HpaII, MspI can cleave the sequence when the internal C residue is methylated.
FAQs
- The NEB catalog has historically stated that activity in NEBuffer4 is less than 100% yet this enzyme is now supplied with NEBuffer4. What have you changed?
- Has the conversion to NEBuffer4 altered any of the properties of the restriction enzyme?
- If I have an old tube of enzyme, what NEBuffer should I use?
- Will the new enzyme work in the originally supplied NEBuffer?
- Why is NEB switching this restriction enzyme to NEBuffer 4?
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 100-fold overdigestion with MspI, > 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, > 95% can be recut with MspI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 500 units of MspI 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 300 units of MspI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.2% of the total radioactivity.
Msp I: DNA cocrystal (Lydia Dorner and Ira Schildkraut, New England Biolabs)
Reagents Sold Separately
NEBuffer 4
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 5,196,331
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