Product Class: Restriction Endonuclease

SphI-HF®
neb31 cloned at NEB recombinant engineered time saver dil_B 37 65 Heat

SphI-HF has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10141297. Learn more.

We are excited to announce that all reaction buffers are now BSA-free. NEB began switching our BSA-containing reaction buffers in April 2021 to buffers containing Recombinant Albumin (rAlbumin) for restriction enzymes and some DNA modifying enzymes. Find more details at www.neb.com/BSA-free.


NEB high fidelity restriction endonuclease that recognizes the sequence GCATG^C
Isoschizomers | Single Letter Code

Product Introduction

High-Fidelity (HF®) restriction enzymes have the same specificity as native enzymes, but have been engineered for significantly reduced star activity and performance in a single buffer (rCutSmart™ Buffer). All HF-restriction enzymes come with Gel Loading Dye, Purple (6X).  Enjoy the enhanced performance and added value of our engineered enzymes at the same price as the native enzyme:

  • Engineered for improved performance
  • 100% activity in rCutSmart Buffer
  • Time-Saver™ qualified for digestion in 5-15 minutes
  • Reduced star activity
  • Supplied with 1 vial of Gel Loading Dye, Purple (6X)
  • Restriction Enzyme Cut Site: GCATG/C
Catalog # Size Concentration
R3182S 500.0 units 20000 units/ml
R3182L 2500.0 units 20000 units/ml
R3182M 2500.0 units 100000 units/ml

Featured Videos

View Video Library
  • ReduceStarActivity_video_thumb

    Reduce Star Activity with High-Fidelity Restriction Enzymes

  • timesaverprotocol_video_thumb

    TIME-SAVER™ Protocol for Restriction Enzyme Digests

  • NEBTVEp15VLthumb280x210

    NEB® TV Ep. 15 – Applications of Restriction Enzymes

  • cuttingclose_video_thumb

    Restriction Enzyme Digest Protocol: Cutting Close to DNA End

  • dnasmear_video_thumb

    Restriction Enzyme Digestion Problem: DNA Smear on Agarose Gel

  • EnzymeNotCutting_video_thumb

    Why is My Restriction Enzyme Not Cutting DNA?

  • TooManyBands_video_thumb

    Restriction Enzyme Digest Problem: Too Many DNA Bands

  • nebcloner_dd_demo_thumb

    Double Digestion with NEBcloner

Product Information

Description

High Fidelity (HF) Restriction Enzymes have 100% activity in rCutSmart Buffer; single-buffer simplicity means more straightforward and streamlined sample processing. HF enzymes also exhibit dramatically reduced star activity. HF enzymes are all Time-Saver 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)
This product is related to the following categories:
Restriction Endonucleases S Products,
High-Fidelity (HF®) Restriction Endonucleases Products,
Time-Saver Qualified Restriction Enzymes Products,
This product can be used in the following applications:
Fast Cloning: Accelerate your cloning workflows with reagents from NEB,
Restriction Enzyme Digestion

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & 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 rCutSmart™ Buffer
Incubate at 37°C

1X rCutSmart™ Buffer
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 µg/ml Recombinant Albumin
(pH 7.9 @ 25°C)

Activity in NEBuffers

NEBuffer™ r1.1: 50%
NEBuffer™ r2.1: 25%
NEBuffer™ r3.1: 10%
rCutSmart™ Buffer: 100%

Diluent Compatibility

Storage Buffer

10 mM Tris-HCl
200 mM NaCl
0.1 mM EDTA
1 mM DTT
200 µg/ml Recombinant Albumin
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

65°C for 20 minutes

Methylation Sensitivity

dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Not Sensitive

Isoschizomers

PaeI
SphI

Product 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.
  3. Not sensitive to CpG, dcm, or dam methylation.

Protocols, Manuals & Usage

Protocols

  1. Optimizing Restriction Endonuclease Reactions
  2. Protocol for Direct Digestion of gDNA during droplet digital PCR (ddPCR)
  3. Restriction Digest Protocol
  4. Protocol for Direct Digestion of gDNA during droplet digital PCR (ddPCR)
  5. Double Digest Protocol with Standard Restriction Enzymes

Usage & Guidelines

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. Is there a difference in cutting close to the ends between SphI-HF and SphI?
  2. How does the level of star activity of SphI-HF compare to SphI?
  3. Why does the HF version of the enzyme have a different recommended buffer than the wild type enzyme?
  4. When should I choose the HF version of the enzyme?
  5. When is star activity a concern?
  6. When should I choose the High Fidelity (HF®) version of the enzyme?
  7. Can the change in buffer preference of the HF enzyme be advantageous?
  8. Will the HF enzyme produce elevated star activity when used in a buffer other than the one recommended?
  9. What does it mean to be Time-Saver™ qualified?
  10. How is the improvement in fidelity of HF restriction endonucleases quantitated?
  11. What is the Fidelity Index (FI)?
  12. What does HF® refer to following the name of a restriction enzyme?
  13. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
  14. 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?
  15. 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?
  16. Can Gel Loading Dye, Purple 6X (B7024) be stored in cold temperatures?
  17. Which NEB restriction enzymes are supplied with Gel Loading Dye, Purple (6X)?
  18. Is this enzyme sensitive to dam, dcm or mammalian CpG methylation?
  19. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?

Troubleshooting

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specification Change Notifications

SphI-HF has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10141297. All subsequent (higher number) lots will contain rAlbumin. For additional information, please visit at NEB Restriction Enzyme formulations with Recombinant Albumin (rAlbumin).

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]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

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.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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