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M0265L |
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1,250 units
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5,000 units/ml |
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M0265S |
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250 units
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5,000 units/ml |
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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 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).
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 nucleotides 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
Notes
General notes:- Exo T has different activity on RNA vs. DNA. For RNA, 1 unit of Exo T is required to completely digest 1.0 pmole of rA20 under standard reaction conditions as mesaured by gel electrophoresis.
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:
Free of detectable endonucleases and 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
- 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.
- Viswanathan, M., Dower, K. D. and Lovett, S. T. (1998) J. Biol. Chem, 273, 35126-35131.
- Zuo, Y. and Deutscher, M. P. (1999) Nucleic Acid Res, 260, 7067-7071.
- Zeng, Y. and Cullen, B. R. (2004) Nucleic Acid Res, 32, 4776-4780.
Reagents Sold Separately
NEBuffer 4
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