Product Source

An E. coli strain that carries the Tsp509I gene from Thermus species (ITI 346).

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 65°C in a total reaction volume of 50 µl.

Reaction Conditions

1X NEBuffer 1
Incubate at 65°C

1X NEBuffer™ 1:
10 mM Bis-Tris-Propane-HCl
10 mM MgCl2
1 mM DTT
pH 7 @ 25°C

Usage Concentration


Activity in NEBuffers

NEBuffer 1: 100%
NEBuffer 2: 100%
NEBuffer 3: 100%
NEBuffer 4: NR

Diluent Compatibility

Storage Conditions

10 mM Tris-HCl
50 mM KCl
10 mM MgCl2
1 mM DTT
0.1 mM EDTA
200 μg/ml BSA
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation


Methylation Sensitivity

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

Activity at Temperature

@37°C: 10%


  1. Tsp509I is sensitive to methylation by EcoRI Methylase.
  2. For enzymes that cannot be heat-inactivated, we recommend using a column for cleanup (such as the Monarch® PCR & DNA Cleanup Kit), or running the reaction on an agarose gel and then extracting the DNA (we recommend Monarch Gel Extraction Kit), or performing a phenol/chloroform extraction.


  1. Optimizing Restriction Endonuclease Reactions
  2. Double Digest Protocol with Standard Restriction Enzymes

Selection Charts

Usage Guidelines & Tips

Interactive Tools

Quality Control

Quality Assurance Statement

  • Tsp509I has been purified free of exonucleases and endonucleases by Finnzymes Oy (Finland), a strategic partner of New England Biolabs, Inc. Quality controls are performed at Finnzymes Oy and confirmed at New England Biolabs, Inc.

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.
  • 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.

Safety Data Sheet

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