Product Class: Nuclease

Exonuclease V (RecBCD)

cloned at neb recombinant NEBuffer 4 incubation temp heat inactivation
Catalog #SizeConcentration
M0345S1,000 units10,000 units/ml
M0345L5,000 units10,000 units/ml

Description

Exonuclease V, a RecBCD complex from E. coli has several different enzyme activities, including an ATP-dependent single-stranded DNA endonuclease activity, ss- and ds- DNA exonuclease activity. The hydrolysis in each case is bi-directional (from both the 3´ and 5´ ends) and processive, producing oligonucleotides (1,2,3). All Exonuclease V activities have divalent cation requirements. Mg2+ is required for the exonuclease activity, while Ca2+ inhibits the exonuclease activity and allows double-stranded DNA unwinding (helicase activity) without hydrolysis (4,5).

Product Source

An E. coli strain containing plasmids for expressing the three subunits of E. coli Exonuclease V: RecB, RecC and RecD.

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
NEBuffer 4-2010X
Adenosine 5'-Triphosphate (ATP)-20*10 mM

* Reagents' Storage Notes

  • Adenosine 5'-Triphosphate (ATP):

    For long term storage (>30 days), store at -70°C.

Advantages and Features

Applications

Degradation of linear ssDNA and dsDNA while preserving nicked and supercoiled plasmid DNA.

Properties and Usage

Unit Definition

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

Reaction Conditions

1X NEBuffer 4
Supplement with 1 mM ATP
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

70°C for 30 min

Unit Assay Conditions

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

Heat Inactivated

Yes

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.
  • Endonuclease Activity (Nicked Double Stranded DNA):
    The product is tested in a reaction containing a nicked DNA substrate. After incubation for 4 hours the percent cleaved is determined by agarose gel electrophoresis.
  • 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.
  • RNase Activity (1 Hour Digestion):
    The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

Supporting Documents

Material Safety Datasheets

The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

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.

References

  1. Eichler, D.C. et al. (1977). J.Biol. Chem. 252, 499-503.
  2. Tayler, A.F. et al. (1985). J. Mol. Biol. 185, 431-443.
  3. Amundsen, S.K. et al. (1986). Proc. Natl. Acad. Sci. 83, 5558-5562.
  4. Palas, K.M. et al. (1990). J. Biol. Chem. 265, 3447-3454.
  5. Dillingham, M.S. et al. (2008). Microbiology and Mol. Biol. Review. 72, 642-671.
  1. A Typical Exonuclease V Reaction (M0345)