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Restriction Endonucleases >
High Fidelity (HF) Restriction Enzymes >
SacI-HF™ |  | | | | SacI-HF™ | | | 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:
New England Biolabs provides customers with high quality tools for a wide range of molecular biology applications. As part of our ongoing commitment to the study and improvement of restriction enzymes, we are pleased to introduce a line of restriction enzymes that have been engineered for maximum performance, convenience and flexibility. These engineered enzymes have the same specificity as their established counterparts with the benefit of reduced star activity. In addition, all of these engineered enzymes work optimally in NEBuffer 4, which has the highest level of enzyme compatibility, and will simplify double digest reactions. They are all Time-Saver qualified, and digest substrate DNA in five minutes. In order to distinguish these engineered enzymes, the letters –HF™ have been added to the restriction enzyme name and they are packaged in unique purple-capped tubes.
Source:
An E. coli strain that carries the cloned and modified (Q117H/R200A) SacI gene from Streptomyces achromogenes (ATCC 12767)
Reagents Supplied:
NEBuffer 4 (10X)
BSA (100X)
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 | |
More information about: Methylation Sensitivity |
Heat Inactivation:
65°C for 20 minutes
Survival in a Reaction:
(+ + +) Suitable for an extended or overnight digestion. Enzyme is active > 8 hours.
More information about: Extended Digests with Restriction Enzymes
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 (HindIII digest) 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 (HindIII digest)
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 @ -20°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent A
Notes
General notes:- SacI-HF is inhibited by salt concentrations above 50 mM. Mini-prep DNA containing residual salt may be resistant to cleavage. A 70% alcohol wash or dialysis can be used to remove the salt.
- SacI-HF is sensitive to cytosine methylation at GAGmCTC but not GAGCTmC and insensitive to adenine methylation at GmAGCTC.
Usage notes:- SacI-HF™ has the same specificity as SacI (NEB #R0156), but it has been 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)?
- How does the level of star activity of SacI-HF compare to SacI?
- What is the difference between SacI-HF and SacI?
- What is the specific activity of SacI-HF?
- Is there any difference in the methylation sensitivity between SacI-HF and SacI?
- Is there a difference in cutting close to the ends between SacI-HF and SacI?
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 SacI-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 SacI-HF™.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 100 units of SacI-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 SacI-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 < 0.05% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 60 units of SacI-HF™ 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
Companion Products
SacI
Legal
Patents:
PCT Publication No: WO2009/009797
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England Biolabs, Inc. is an ISO 9001 and ISO
14001 Certified Company.
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