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SphI-HF®
High Fidelity Enzymes
Product Update: A free vial of Gel Loading Dye, Purple (6x) is now included with all HF restriction enzymes.
Isoschizomers | Single Letter Code | Icon Descriptions
Catalog #
R3182S
Size
500 units
Concentration
20,000 units/ml
Price
$66.00
Qty
R3182L
2,500 units
20,000 units/ml
$267.00
1
R3182M
2,500 units
100,000 units/ml
$267.00
1
1
Categories: High-Fidelity (HF®) Restriction Endonucleases, Restriction Endonucleases: S, Time-Saver™ Qualified Restriction Enzymes
Applications: Restriction Enzyme Digestion
Product
Information
FAQs &
Tech Tips
Protocols &
Manuals
Other Tools &
Resources
Description
Related Products
Quality &
Safety
Legal
Information
Properties and Usage
Notes
Description
High Fidelity (HF®) Restriction
Enzymes have 100% activity in
CutSmart™ Buffer; singlebuffer simplicity means more
straightforward and streamlined sample processing. HF enzymes also exhibit dramatically reduced star activity. HF enzymes are all TimeSaver™ qualified and can therefore cut substrate DNA in 5-15 with the flexibility to digest overnight without degradation to DNA. Engineered
with performance in mind, HF restriction enzymes are fully active under a broader range of conditions, minimizing off-target products, while
offering flexibility in experimental design.
NEB extensively performs quality controls on all standard and high-fidelity (HF®) restriction enzymes. Examples of nuclease contamination
studies for some of our HF restriction enzymes are shown below.
Restriction Enzyme Competitor Study: Nuclease Contamination
EcoRI, NotI, and BamHI from multiple suppliers were tested in reactions containing a fluorescent labeled single stranded, double stranded
blunt, 3’overhang or 5’ overhang containing oligonucleotides. The percent degradation is determined by capillary electrophoresis and peak
analysis. The resolution is at the single nucleotide level.
Product Source
An E. coli strain that carries the cloned and modified SphI gene from Streptomyces phaeochromogenes (NRRL B-3559)
Reagents Supplied
The following reagents are supplied with this product:
Store at (°C)
Concentration
CutSmart® Buffer
-20
10X
Gel Loading Dye, Purple (6X)
25
6X
Properties and Usage
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.
Reaction Conditions
1X CutSmart® Buffer
Incubate at 37°C
1X CutSmart® Buffer:
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 μg/ml BSA
pH 7.9 @ 25°C
Activity in NEBuffers
NEBuffer 1.1: 50%
NEBuffer 2.1: 25%
NEBuffer 3.1: 10%
CutSmart® Buffer: 100%
Diluent Compatibility
Diluent B
Storage Conditions
10 mM Tris-HCl
200 mM NaCl
0.1 mM EDTA
1 mM DTT
200 μg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Heat Inactivation
65°C for 20 min
Methylation Sensitivity
dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Not Sensitive
Related Products
Companion Products
SphI
Monarch® Plasmid Miniprep Kit
Monarch® DNA Gel Extraction Kit
Monarch® PCR & DNA Cleanup Kit (5 μg)
Materials Sold Separately
CutSmart® Buffer
Gel Loading Dye, Purple (6X)
Notes
1. Cleaves to leave a 3' CATG extension which can be efficiently ligated to DNA fragments generated by NlaIII.
2. Based on the stability of the enzyme in the reaction, incubations longer than 1 hr will not result in improved digestion, unless additional
enzyme is added. Please refer to Restriction endonuclease survival in a reaction for more information regarding this topic.
FAQs
FAQs
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Is there a difference in cutting close to the ends between SphI-HF and SphI?
How does the level of star activity of SphI-HF compare to SphI?
Why does the HF version of the enzyme have a different recommended buffer than the wild type enzyme?
When should I choose the HF version of the enzyme?
When is star activity a concern?
When should I choose the High Fidelity (HF®) version of the 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)?
What does HF® refer to following the name of a restriction enzyme?
What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
How can I access the old NEBuffer Activity Chart?
I tested your restriction enzyme on the substrate DNA recommended by NEB, and it appears to be active, however it does not
digest my DNA. What could be the reason?
Is Gel Loading Dye, Purple (6X) or Gel Loading Dye, Purple (6X), no SDS compatible with other DNA binding dyes such as
SYBR® and GelRed™ during gel electrophoresis?
Can Gel Loading Dye, Purple 6X (B7024) be stored in cold temperatures?
Why do I see additional DNA bands on my gel after a restriction digest?
Why is my Restriction Enzyme not cutting DNA?
Why do I see a DNA smear on an agarose gel after a restriction digest?
How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?
Protocols
Protocols
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Optimizing Restriction Endonuclease Reactions
Double Digest Protocol with Standard Restriction Enzymes
Time-Saver Protocol for Restriction Enzyme Digests
Protocol for Direct Digestion of gDNA during droplet digital PCR (ddPCR)
Protocol for Digestion Prior to droplet digital PCR (ddPCR)
Selection Charts
Troubleshooting Guides
Selection Charts
Alphabetized List of Recognition Specificities
Usage Guidelines & Tips
Interactive Tools
Buffer and Diluent Formulation Table
Compatible Cohesive Ends and Generation of New Restriction Sites
Cross Index of Recognition Sequences
Dam-Dcm and CpG Methylation
Frequencies of Restriction Sites
Isoschizomers
Time-Saver™ Qualified Enzymes
Why Choose Recombinant Enzymes?
Usage Guidelines & Tips
Activity at 37°C for Restriction Enzymes with Alternate Incubation Temperatures
Activity of Restriction Enzymes in PCR Buffers
Cleavage Close to the End of DNA Fragments
Cleavage of Supercoiled DNA
Digestion of Agarose-Embedded DNA: Info for Specific Enzymes
Double Digests
Effects of CpG Methylation on Restriction Enzyme Cleavage
Heat Inactivation
Megabase Mapping
NEBuffer Activity/Performance Chart with Restriction Enzymes
Optimizing Restriction Endonuclease Reactions
Reduced Star Activities of HF® Enzymes
Restriction Endonucleases - Survival in a Reaction
Restriction Enzyme Diluent Buffer Compatibility
Restriction Enzyme Tips
Restriction Enzymes for Droplet Digital PCR (ddPCR)
Single Letter Codes
Star Activity
Traditional Cloning Quick Guide
Troubleshooting Guides
Restriction Enzyme Troubleshooting Guide
Interactive Tools
Competitor Cross-Reference Tool
DNA Sequences and Maps Tool
Double Digest Finder
Enzyme Finder
NEBcutter®
NEBioCalculator
REBASE®
Quality Control
Specifications
Certificate of Analysis
Safety Data Sheet
Quality Control
Quality Control Assays
The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product.
Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting
Documents section of this page. Further information regarding NEB product quality can be found here.
Blue-White Screening (Terminal Integrity):
A sample of DNA vector linearized with a 10-fold excess of a restriction endonuclease, religated and transformed into an E. coli
strain expressing the LacZ beta fragment gene results in less than 1% white colonies.
Endonuclease Activity (Nicking):
The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to
the nicked form is determined by agarose gel electrophoresis.
Exonuclease Activity (Radioactivity Release):
The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4
hours the exonuclease activity is determined by the % release of radioactive nucleotides.
Ligation and Recutting (Terminal Integrity):
After an over-digestion of DNA with a restriction endonuclease the percentage of the DNA fragments ligated with T4 DNA ligase
and the percentage that can be recut are determined by agarose gel electrophoresis.
Non-Specific DNase Activity (16 hour):
The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16
hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
Certificate of Analysis
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's
for an individual lot. The following file naming structure is used to name these document files: [Product
Number]_[Size]_[Version]_[Lot Number]
R3182M_v1_0011212
R3182M_v1_0011304
R3182M_v1_0011310
R3182S_L_v1_0011212
R3182S_L_v1_0011304
R3182S_L_v1_0011310
R3182S_L_v1_0011403
R3182M_v1_0011403
R3182M_v1_0011409
R3182S_L_v1_0011409
R3182S_L_v1_0031504
R3182M_v1_0031506
R3182S_L_v1_0031509
R3182M_v1_0031512
R3182S_L_v1_0031512
R3182S_L_v1_0031608
R3182M_v1_0031611
R3182S_L_v1_0031701
R3182M_v1_0031706
Specifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the
product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
R3182M_v1
R3182S_L_v1
Safety Data Sheet
The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
SphI-HF®
CutSmart® Buffer
Gel Loading Dye, Purple (6X)
Legal and Disclaimers
Legal and Disclaimers
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc
(NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain
additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes
in humans or animals.