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DNA Modifying Enzymes and Cloning >
Nucleases >
Exonuclease T |
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Prices are in US dollars and valid only for US orders.
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- Isolated from a recombinant source
- Supplied with 10X Reaction Buffer
- Specific for single-stranded DNA or RNA
Description:
Exonuclease T (Exo T), also known as RNase T, is a single-stranded DNA (1,2) or RNA (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 a DNA molecule that has a 3' extension (2). The efficiency of this reaction is sequence dependent (5) and may result in a mixture of products having 1 or 2 base 3' recessed ends as well as blunt ends (5).
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:
NEBuffer 4 (10X)
Enzyme Properties
Heat Inactivation:
65°C for 20 minutes
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 4
Incubate at
25°C.
1X NEBuffer 4:
20 mM Tris-acetate
50 mM potassium acetate
10 mM Magnesium Acetate
1 mM Dithiothreitol
pH 7.9 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to produce 0.1 nmol of TCA soluble DNA from 1 nmol of [3H]-labeled polythymidine in a total reaction volume of 100 μl in 30 minutes at 25°C in 1X NEBuffer 4 with 1 nmol [3H]-labeled polythymidine DNA.
Concentration:
5,000 units/ml
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
FAQs
- Will Exonuclease T degrade single stranded RNA?
- Can Exonuclease T be heat inactivated?
- What is the activity of Exonuclease T in the NEBuffers?
- What is the molecular weight of Exonuclease T?
- Does Exonuclease T work as well as DNA Polymerase I, Large (Klenow) Fragment (NEB# M0210) for 3' extension removal?
- How is Exonuclease T different from Exonuclease I (NEB# M0293)?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Purified free of endonucleases and contaminating exonucleases.
5' to 3' Exonuclease Activity:
No detectable 5' → 3' nuclease activity was observed when
10 units of Exonuclease T was incubated with substrates containing either 5' extensions or blunt ends.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 10 units of Exonuclease T with 1 μg of
ΦX174 RF I DNA for 4 hours at 25ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
References
- Viswanathan, M., Dower, K.D. and Lovett, S.T. (1998) J. Biol. Chem., 273, 35126-35131.
- Zuo, Y. and Deutscher, M.P. (1999) Nucl. Acids Res., 27, 4077-4082.
- Deutscher, M.P., Marlor, C.W. and Zaniewski, R. (1984) Proc. Natl. Acad. Sci. USA, 81, 4290-4293.
- Deutscher, M.P. and Marlor, C.W. (1985) J. Biol. Chem., 260, 7067-7071.
- Whitaker, R., unpublished observations.
Reagents Sold Separately
NEBuffer 4
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