Home DNA Modifying Enzymes and Cloning Technologies Exonuclease T

Exonuclease T
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Exonuclease I (E.coli), Thermostable Exonuclease I, Exonuclease T mechanism


  • Also known as RNase T
  • Single-stranded (ssDNA or RNA) specific exonuclease
  • Catalyzes the removal of nucleotides from linear single-stranded DNA or RNA in the 3' to 5' direction
  • Need help finding the right exonuclease for your experiments? Try Exo Selector 

Exonuclease T can be used for:

  • Removal of 3' overhangs of double-stranded DNA to generate blunt-ends*
  • Removal of single-stranded primers in PCR reactions prior to DNA sequencing or SNP analysis
  • Removal of single-stranded primers for nested PCR reactions
  • Removal of single-stranded DNA, leaving behind double-stranded DNA in the sample
  • Not sure which RNase to choose? Learn more about the different RNases offered by NEB.

*sequence-dependent

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Exo_Intro_Anim_Thumb_720

What are exonucleases and their applications?

Catalog # Cat # Concentration Conc Size List Price List Price Your Price Quantity
M0265S 5,000 units/ml 250 units $88.00 $88.00
M0265L 5,000 units/ml 1,250 units $358.00 $358.00
Please enter a quantity for at least one size
  • 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 (4).

    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).
    This product is related to the following categories:
    RNases,
    Epitranscriptome Analysis,
    Exonucleases and Non-specific Endonucleases

    • Reagents Supplied

      The following reagents are supplied with this product:

      NEB # Component Name Component # Stored at (°C) Amount Concentration

    • Properties & Usage

      Unit Definition

      One unit is defined as the amount of enzyme required to release 1 nmol of single dT nucleotides from 50 pmol of Fam-labeled polythymidine substrate in 30 minutes at 25°C.

      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)

      Activity in NEBuffers

      NEBuffer™ 1: 100%
      NEBuffer™ 2: 75%
      NEBuffer™ 3: 25%

      Storage Buffer

      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 minutes

    • Related Products

      Materials Sold Separately

    • Product 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.
      3. Exonuclease T removes ssDNA extensions but does not work well when two cytosine nucleotides are in a row.  “Although RNase T appears to bind up to 10 nucleotides in its active site, its specificity is defined largely by the last 4 residues. A single 3'-terminal C residue can reduce RNase T action by >100-fold, and 2-terminal C residues essentially stop the enzyme.” (PMID: 12050169 DOI: 10.1074/jbc.M204252200)

    • 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.

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