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Restriction Endonucleases >
Restriction Endonucleases >
BstEII |
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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 BstEII gene from Bacillus stearothermophilus ET (N. Welker).
Reagents Supplied:
NEBuffer 3 (10X)
BSA (100X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 50% | | NEBuffer 2: | | 75% | | NEBuffer 3: | | 100% | | NEBuffer 4: | | 75% |
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 |
Activity at 37°C:
50%
Heat Inactivation:
No
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 3
Supplemented with 100 μg/ml Bovine Serum Albumin
Incubate at
60°C.
1X NEBuffer 3:
50 mM Tris-HCl
100 mM NaCl
10 mM MgCl2
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 60°C in a total reaction volume of 50 µl.
Concentration:
10,000 units/ml and 50,000 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:- Conditions of low ionic strength, high enzyme concentration, glycerol concentration > 5%, or pH > 8.0 may result in star activity.
- Of the over 3,000 known restriction endonucleases, BstEII is one of the few that produces extensions of more than 4 bases.
Usage notes:- Due to potential for star activity, it is recommended that extended incubations be done at 37°C .
FAQs
- Do degenerate recognition sites need to be palindromic?
- How can this enzyme be inactivated?
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 BstEII, > 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 BstEII.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 50 units of BstEII incubated for 16 hours at 60ºC
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 50 units of BstEII with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 60ºC
released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 25 units of BstEII with 1 μg of
ΦX174 RF I DNA for 4 hours at 60ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
Reagents Sold Separately
NEBuffer 3
BSA
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