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DNA Modifying Enzymes and Cloning >
DNA Repair Proteins >
T7 Endonuclease I |
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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
- New wildtype T7 Endonuclease I has higher cleavage efficiency to mismatched DNA
- Recognizes non-perfectly matched DNA
Description:
T7 Endonuclease I recognizes and cleaves non-perfectly matched DNA, cruciform DNA structures, Holliday structures or junctions, heteroduplex DNA and more slowly, nicked double-stranded DNA. The cleavage site is at first, second or third phosphodiester bond that is 5´ to the mismatch. The protein is the product of T7 gene 3.
Source:
A fusion of maltose binding protein and truncated, inactive T7 Endonuclease I (tT7 Endo I) possessing a C-terminal thioester is purified to near homogeneity using the IMPACT-TWIN system. The full-length, active T7 Endo I is generated in vitro by ligating a synthetic peptide, consisting of the truncated amino acid residues, to the thioester-tagged tT7 Endo I (1).
Applications:- Resolve four-way junction or branched DNA
- Detect or cleave heteroduplex and nicked DNA
- Randomly cleave linear DNA for shot-gun cloning
Reagents Supplied:
NEBuffer 2 (10X)
pUC(AT)
Enzyme Properties
Heat Inactivation:
No
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 2
Incubate at
37°C.
1X NEBuffer 2:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to convert > 90% of 1 µg of supercoiled cruciform pUC(AT) to > 90% linear form in a total reaction volume of 50 µl in 1 hour at 37°C.
Concentration:
10,000 units/ml
Storage Conditions:
20 mM Tris-HCl
200 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
0.15% Triton X-100
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- pUC(AT) is derived from pUC19 with a modification of the polylinker between the EcoRI site and the PstI site.
Usage notes:- T7 Endonuclease I is a structure-selective enzyme. It acts on a variety of DNA substrates with different specific activities. It is important to control the amount of enzyme and the reaction time used for cleavage of a particular substrate.
- Temperatures above 42°C cause an increase in nonspecific nuclease activity and should be avoided.
FAQs
- Does T7 Endonuclease I recognize single base pair mismatches?
- What is the activity of T7 Endonuclease I in other buffers?
- What is the activity of T7 Endonuclease I on various DNA substrates?
- Does Mn enhance T7 Endonuclease I activity?
- Can T7 Endonuclease I be heat inactivated?
- What is the activity of T7 Endonuclease I at different reaction temperatures?
- What is the molecular weight of T7 Endonuclease I?
- What common additives inhibit T7 Endonuclease I?
- How stable is T7 Endonuclease I in reaction?
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 contaminating exonucleases and endonucleases.
Ligation:
16 µg of HaeIII digested ΦX174 DNA was incubated with 40 units of T7 Endonuclease I in a total reaction volume of 400 µl for 2 hours at 37°C. The DNA was ethanol precipitated and ligated with T4 DNA Ligase and then recut with HaeIII. Approximately 50% of the T7 Endonuclease I treated fragments were ligated and of those, > 95% were recut with HaeIII.
References
- Xu, M. -Q. and Evans, T.C. (2001) Methods, 24, 257-277.
- Parkinson, M.J. and Lilley. D.M.J. (1997) J. Mol. Biol., 270, 169-178.
- White, M.F. et al. (1997) J. Mol. Biol., 269, 647-664.
- Hadden, J.M. et al. (2001) Nat. Struct. Biol., 8, 62-67.
Reagents Sold Separately
NEBuffer 2
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