T5 Exonuclease

Description

T5 Exonuclease degrades DNA in the 5´ to 3´ direction (1). T5 Exonuclease is able to initiate nucleotide removal from the 5´ termini or at gaps and nicks of linear or circular dsDNA (1). However, the enzyme does not degrade supercoiled dsDNA (2). T5 Exonuclease also has ssDNA endonuclease activity.

Product Source

An E. coli strain that carries a plasmid with the T5 phage D15 gene.

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
NEBuffer 4-2010X

Advantages and Features

Applications

  • Degradation of linear ssDNA, dsDNA or nicked plasmid DNA while maintaining supercoiled plasmid DNA.
  • Remove denatured DNA isolated during alkaline lysis-based plasmid purification procedure (5).
  • Enhance the transfection efficiency of miniprep from plasmid cDNA libraries (6).

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme required to produce 1 nmol of acid soluble deoxyribonucleotide from double-stranded DNA in 30 minutes at 37°C in a total reaction volume of 50 μl.

Reaction Conditions

1X NEBuffer 4
Incubate at 37°C

1X NEBuffer 4:
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
1 mM DTT
pH 7.9 @ 25°C

Storage Temperature

-20°C

Storage Conditions

50 mM Tris-HCl
100 mM NaCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
0.1% Triton® X-100
pH 7.5 @ 25°C

Heat Inactivation

No

Unit Assay Conditions

1X NEBuffer 4, 0.15 mM sonicated duplex [3H]-DNA.

References

  1. Sayers, J.R. et al. (1991). Nucleic Acids Res. 19, 4127-4132.
  2. Sayers, J.R. et al. (1990). J. of Biol. Chem. 265, 18311-18317.
  3. Kaliman, A.V. et al. (1986). FEBS. 195, 61-64.
  4. Frenkel, G.D. et al. (1971). J. of Biol. Chem. 246, 4839-4847.
  5. Sayers, J.R. et al. (1996). Analytical Biochem. 241, 186-189.
  6. Kiss-Toth, E. et al. (2001). Analytical Biochem. 288, 230-232.

FAQs

  1. How Does T5 Exonuclease differ from Lambda Exonuclease (NEB# M0262)?
  2. Will T5 exonuclease cleave supercoiled dsDNA?
  3. How does T5 Exonuclease differ from Exonuclease III (NEB# M0206)?
  4. What is the activity of T5 Exonuclease in the NEBuffers?
  5. Can T5 Exonuclease be heat inactivated?
  6. Can DNA be blunted using T5 Exonuclease?
  7. Will T5 Exonuclease work in CutSmart buffer?

Tech Tips

Has 5’ to 3’ dsDNA exonuclease activity and ssDNA endonuclease activity.

Protocols

  1. Protocol for T5 Exonuclease (M0363)

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Selection Charts

Interactive Tools

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.
  • 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.
  • Protein Purity (SDS-PAGE):
    The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.

Safety Data Sheet

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

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.