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Restriction Endonucleases >
Restriction Endonucleases >
SphI-HF™ |  | | | | SphI-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
Description:
Many restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. This altered or relaxed specificity has been termed star activity. HF restriction endonucleases have been engineered to cleave with higher fidelity than the wild type enzyme, hence exhibiting less star activity.
Source:
An E. coli strain that carries the cloned and modified (K100A) SphI gene from Streptomyces phaeochromogenes (NRRL B-3559)
Reagents Supplied:
NEBuffer 4
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 50% | | NEBuffer 2: | | 25% | | 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:
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
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
Unit Assay Substrate:
λ DNA
Storage Conditions:
10 mM Tris-HCl
200 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
100 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent B
Notes
General notes:- Cleaves to leave a 3' CATG extension which can be efficiently ligated to DNA fragments generated by NlaIII.
Usage notes:- SphI-HF™ has the same specificity as SphI (NEB #R0182), but it has been engineered for reduced star activity.
FAQs
- What is the specific activity of SphI-HF™?
- What does HF refer to following the name of the restriction enzyme?
- 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 is the difference between SphI-HF and SphI?
- When should I choose the HF version of the enzyme?
- When is star activity a concern?
- Is there a difference in cutting close to the ends between SphI-HF and SphI?
- What does it mean to be Time-Saver qualified?
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 20-fold overdigestion with SphI-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 SphI-HF™.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 25 units of SphI-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 SphI-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 SphI-HF™ with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 20% 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. 5,262,318
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