PstI has an HF version supplied with CutSmart Buffer. Now comes supplied with 1 vial of Gel Loading Dye, Purple (6X)
cloned at neb recombinant timesaver 5min icon_neb3_1 dil_C-grey incubation temp heat inactivation blue white
Catalog #SizeConcentrationPriceQtyAdd to Cart
R0140S10,000 units20,000 units/ml$63.00Add to Cart
R0140T10,000 units100,000 units/ml$63.00Add to Cart
R0140L50,000 units20,000 units/ml$254.00Add to Cart
R0140M50,000 units100,000 units/ml$254.00Add to Cart
Restriction Endonucleases: P-R,
Time-Saver™ Qualified Restriction Enzymes
BioBrick® Assembly


PstI has a High Fidelity version PstI-HF® (NEB #R3140).

High Fidelity (HF®) Restriction Enzymes have 100% activity in CutSmart™ 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 minutes 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.

Product Source

A E. coli strain that carries the PstI gene from Providencia stuartii 164 (ATCC 49762).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
NEBuffer™ 3.1-2010X

Properties and Usage

Unit Definition

One unit is defined as the amount of PstI 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 NEBuffer 3.1
Incubate at 37°C

1X NEBuffer™ 3.1:
100 mM NaCl
50 mM Tris-HCl
10 mM MgCl2
100 μg/ml BSA
pH 7.9 @ 25°C

Activity in NEBuffers

NEBuffer 1.1: 75%
NEBuffer 2.1: 75%
NEBuffer 3.1: 100%
CutSmart® Buffer: 50%

Diluent Compatibility

Storage Temperature


Storage Conditions

250 mM NaCl
10 mM Tris-HCl
1 mM DTT
0.1 mM EDTA
200 μg/ml BSA
50% Glycerol
0.15% Triton® X-100
pH 7.4 @ 25°C

Heat Inactivation

80°C for 20 min

Methylation Sensitivity

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


  1. May exhibit star activity in CutSmart Buffer.


  1. What is the activity of PstI at 25°C?
  2. Is PstI used in special techniques?
  3. When is star activity a problem for PstI?
  4. What is the molecular weight of PstI?
  5. Is PstI blocked by methylation?
  6. Is PstI inhibited by dUTP incorporated at the site?
  7. Does spermidine increase activity?
  8. Are there specific surrounding sequences that produce slow or resistant sites?
  9. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
  10. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
  11. How can I access the old NEBuffer™ Activity Chart?
  12. Why do I see a DNA smear on an agarose gel after a restriction digest?
  13. Why do I see additional DNA bands on my gel after a restriction digest?
  14. Why is my Restriction Enzyme not cutting DNA?
  15. How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?
  16. Which NEB restriction enzymes are supplied with Gel Loading Dye, Purple (6X)?

Tech Tips

Blocked by some types of methylation often found in plant DNA.


  1. Optimizing Restriction Endonuclease Reactions
  2. Double Digest Protocol with Standard Restriction Enzymes
  3. Time-Saver Protocol for Restriction Enzyme Digests

Selection Charts

Usage Guidelines & Tips

Troubleshooting Guides

Interactive Tools


  • Yang GQ, Chen YM, Wang JP, Guo C, Zhao L, Wang XY, Guo Y, Li L, Li DZ, Guo ZH. (2016). Development of a universal and simplified ddRAD library preparation approach for SNP discovery and genotyping in angiosperm plants. Plant Methods. . Aug 4;12, 39.
  • Guo B, DeFaveri J, Sotelo G, Nair A, Merilä J. (2015). Population genomic evidence for adaptive differentiation in Baltic Sea three-spined sticklebacks. BMCBiol.. Mar 24;13:19,
  • Combosch DJ, Vollmer SV. (2015). Trans-Pacific RAD-Seq population genomics confirms introgressive hybridization in Eastern Pacific Pocillopora corals. Mol Phylogenet Evol. . Jul;88, 154-62.

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.
  • 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]


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]

Safety Data Sheet

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