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Restriction Endonucleases >
Restriction Endonucleases >
PmeI |
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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 PmeI gene from Pseudomonas mendocina (B. Zhou).
Reagents Supplied:
NEBuffer 4
BSA
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 0% | | NEBuffer 2: | | 50% | | NEBuffer 3: | | 10% | | 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 some combinations of overlapping |
Heat Inactivation:
65°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 100 μg/ml Bovine Serum Albumin
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:
10,000 units/ml
Unit Assay Substrate:
λ DNA
Storage Conditions:
10 mM Tris-HCl
100 mM NaCl
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:- PmeI is an octanucleotide-recognizing restriction endonuclease pNEB193, a cloning vector which contains single sites for NEB's 8-base cutters AscI, PacI and PmeI is available (NEB #N3051).
- Four units of PmeI are required for complete digestion of 1 µg of pNEB193.
- For extended digests, incubation at 25°C increases enzyme stability.
FAQs
- What is Star Activity and how can it be avoided?
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 10-fold overdigestion with PmeI, > 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 PmeI.
Digestion of DNA embedded in Agarose: Incubation in reaction buffer, of the DNA containing agarose plug, and Pme I at 4°C for 16 hours followed by 2 hours at 37°C is suggested.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 16 units of PmeI 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 PmeI 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.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 16 units of PmeI with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 5% conversion to RFII as determined by agarose gel electrophoresis.
Blue/White Screening Assay:
An appropriate vector is digested at a unique site within the
lacZα gene with a 10-fold excess of enzyme.
The vector DNA is then ligated, transformed, and plated onto Xgal/IPTG/Amp plates.
Successful expression of β-galactosidase is a function of how intact its gene remains after cloning,
an intact gene gives rise to a blue colony, removal of even a single base gives rise to a white colony.
Enzyme preparations must produce fewer than 3% white colonies to be Blue/White certified.
Reagents Sold Separately
NEBuffer 4
BSA
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 5,945,288
New England Biolabs, Inc.: U.S. Patent No. 5,196,330
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