Exonuclease T

Description

Exonuclease T (Exo T), also known as RNase T, is a single-stranded RNA (1,2) or DNA (3,4) specific nuclease that requires a free 3´ terminus and removes nucleotides in the 3´→ 5´ direction. Exonuclease T can be used to generate blunt ends from RNA (5) or DNA molecules that have 3´ extensions (2).

Highlights

Isolated from a recombinant source

Product Source

Exonuclease T is overexpressed and purified as a C-terminal fusion to maltose-binding protein (MBP). MBP is removed from Exonuclease T by Factor Xa cleavage and Exonuclease T is then purified away from Factor Xa and MBP. Exonuclease T cleaved from MBP has an additional amino acid on the N-terminus and a Phe instead of a Met (i.e., Glu-Phe-Exo T instead of Met-Exo T).

Reagents Supplied

The following reagents are supplied with this product:

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

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme required to produce 0.1 nmol of TCA soluble nucleotides from 1 nmol of [3H]-labeled polythymidine in 1X NEBuffer 4 in 30 minutes at 25°C in a total reaction volume of 100 μl.

Reaction Conditions

1X NEBuffer 4
Incubate at 25°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

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

Heat Inactivation

65°C for 20 min

Notes

  1. Exonuclease T has a different activity on RNA vs. DNA. For RNA, 1 unit of Exonuclease T is required to completely digest 1.0 pmol of rA20 under standard reaction conditions as measured by gel electrophoresis.
  2. Single stranded RNA molecules containing C residues at its 3'-terminus might be difficult to digest with Exonuclease T.

References

  1. Deutscher, M. P., Marlor, C. W. and Zaniewski, R. (1984). Proc. Natl. Acad. Sci. USA. 81, 4290–4293.
  2. Deutscher, M. P. and Marlor, C. W. (1985). J. Biol. Chem. 260, 7067–7071.
  3. Viswanathan, M., Dower, K. D. and Lovett, S. T. (1998). J. Biol. Chem. 273, 35126–35131.
  4. Zuo, Y. and Deutscher, M. P. (1999). Nucleic Acid Res. 27, 4077–4082.
  5. Zeng, Y. and Cullen, B. R. (2004). Nucleic Acid Res. 32, 4776–4780.
  6. Zuo, Y. and Deutscher, M.P. (2002). J. Biol. Chem. 277, 29654-61.

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 Assurance Statement

  • Free of detectable endonucleases and exonucleases.

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.

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.