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Restriction Endonucleases >
Restriction Endonucleases >
BamHI-HF™ |  | | | | BamHI-HF™ | | | New | High-Fidelity Enzymes | |
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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:
An E. coli strain that carries the cloned and modified (E163A/E167T)) BamHI gene from Bacillus amyloliquefaciens H (ATCC 49763)
Reagents Supplied:
NEBuffer 4 (10X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 100% | | 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: Not sensitive |
Heat Inactivation:
No
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 ug of λ DNA in 1 hour at 37°C in a total reaction volume of 50 μl.
Concentration:
20,000 units/ml
Unit Assay Substrate:
λ DNA
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
1 mM Dithiothreitol
200 µg/ml BSA
50% Glycerol
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent A
Notes
Usage notes:- Bam-HI-HF™ has the same specificity as Bam HI (NEB #R0136), but it was engineered for reduced star activity.
FAQs
- What does HF refer to following the name of the restriction enzyme?
- When should I choose the HF version of the enzyme?
- When is star activity a concern?
- Why does the HF version of the enzyme have a different recommended buffer than the wild type enzyme?
- Can the change in buffer preference of the HF enzyme be advantageous?
- Will the HF enzyme produce elevated star activity when used in a buffer other than the one recommended?
- What does it mean to be Time-Saver qualified?
- How is the improvement in fidelity of HF restriction endonucleases quantitated?
- What is the Fidelity Index (FI)?
- Is there a difference in cutting close to the ends between BamHI-HF and BamHI?
- Is there any difference in the methylation sensitivity between BamHI-HF and BamHI?
- How does the level of star activity of BamHI-HF compare to BamHI?
- What is the difference between BamHI-HF and BamHI?
- What is the specific activity of BamHI-HF?
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 15-fold overdigestion with BamHI-HF™, > 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 BamHI-HF™.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 100 units of BamHI-HF™ 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 BamHI-HF™ 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 100 units of BamHI-HF™ with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 10% 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
Legal
Patents:
New England Biolabs, Inc. : U.S. Patent No. 5,137,823
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