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9°N™ DNA Ligase |
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Prices are in US dollars and valid only for US orders.
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- Can be used to ligate nicks in DNA while incubating at high temperatures
- Extremely thermostable and can withstand PCR conditions
- Will not ligate short 4 base overlaps (typical of restriction enzyme
digests), while it efficiently ligates 12 base pair overlaps - Isolated from a recombinant source
Description:
9°N™ DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5´ phosphate and 3´ hydroxyl termini of two adjacent oligonucleotides which are hybridized to a complementary target DNA. 9°N DNA Ligase is active at elevated temperatures (45°C-90°C).
Source:
Purified from an E. coli strain containing the cloned ligase gene from the extremely thermophilic marine archaea Thermococcus sp.(strain 9°N). The archaea was isolated from a submarine thermal vent, at a depth of 2,500 meters, 9° north of the equator at the East Pacific Rise (1).
Applications:- Allele-specific gene detection using Ligase Detection Reaction and Ligase Chain Reaction (2,4).
- Mutagenesis by incorporation of a phosphorylated oligonucleotide during PCR amplification (5).
Reagents Supplied:
9°N DNA Ligase Reaction Buffer (10X)
Enzyme Properties
Heat Inactivation:
No
Reaction & Storage Conditions
Reaction Conditions:
1X 9°N DNA Ligase Reaction Buffer
1X 9°N DNA Ligase Reaction Buffer:
10 mM Tris-HCl
600 µM ATP
2.5 mM MgCl2
2.5 mM Dithiothreitol
0.1 % Triton X-100
pH 7.5 @ 25°C
Unit Definition:
(Cohesive End Unit) One unit is defined as the amount of enzyme required to give 50% ligation of the 12-base pair cohesive ends of 1 µg of BstEII-digested λ DNA in a total reaction volume of 50 µl in 15 minutes at 45°C.
A cohesive end unit is equivalent to the nick-closing unit (1).
Unit Assay Conditions: 1X 9°N DNA Ligase Reaction Buffer and 20 µg/ml BstEII-digested λ DNA in a 50 µl reaction. After incubation at 45°C for 15 minutes, the reaction is terminated by addition of stop dye (50% glycerol, 50 mM EDTA and bromophenol blue), heated at 70°C for 10 minutes and then loaded on a 0.7% agarose gel. Due to the presence of ligase, the cos ends of BstEII-digested λ DNA will stay together after 70°C heat treatment.
Concentration:
40,000 units/ml
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
10 mM ammonium sulfate
Storage Temperature:
-20°C
Notes
Usage notes:- 9°N DNA Ligase is not a substitute for T4 DNA Ligase. The cohesive end unit is equivalent to the nick-closing unit of Barany et al.
- Incubate DNA and enzyme in 1X 9°N DNA Ligase Reaction Buffer at 45°C for 15 minutes or in a thermocycler with a program suited to the reaction described by Barany (1991) Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase. Proc. Natl. Acad. Sci. USA 88, 189-193. The reaction is stopped with a mixture of 50% glycerol, 50 mM EDTA, bromphenol blue.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Phosphatase Activity:
Incubation of 5,000 units for 16 hours at 37°C in NEBuffer 2 released less than 0.05 µmol of inorganic phosphate from para-nitrophenol phosphate.
Non-specific Endonuclease and Exonuclease Activity:
Incubation of 5,000 units for 16 hours at 37°C in NEBuffer 2 with 1 µg 2-Log DNA Ladder fragments does not alter the expected banding pattern on agarose gels.
References
- Thermococcus sp. (strain 9°N-7) isolated by Dr. Holger Jannasch (1991) , Woods Hole Oceanographic Institute.
- Barany, F. (1991) Proc. Natl. Acad. Sci. USA, 88, 189-193.
- Takahashi, M. et al. (1984) J. Biol. Chem., 259, 10041?10047.
- Barany, F. (1991) The Ligase Chain Reaction in a PCR World, (pp. 5-16), Cold Spring Harbor: Cold Spring Harbor Laboratory.
- Michael, S.F. (1994) Biotechniques, 16, 411-412.
Legal
Research Use Assurance:
Research Use Only - No License to Use in Any Patented Process Granted or Implied Notice to Purchaser about Limited License
This product is designed to ligate DNA fragments at temperatures requiring a thermoactive and thermostable enzyme. The seller is aware that the product may be used in the Ligase Chain Reaction (LCR™) process covered by one or more claims of a pending patent application or issued patent assigned to Cornell Research Foundation, Inc. or Cornell Research Foundation, Inc. and the California Institute of Technology. The seller has a limited license under such patent rights, and purchase of this product includes a fully paid-up limited, non-exclusive sublicense under such intellectual property rights to use this product to perform the Ligase Chain Reaction™ process only for research and development activities and for industrial quality assurance testing. No other license has been granted expressly, impliedly or by estoppel to seller, and seller grants no other license or sublicense expressly, impliedly or by estoppel.
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