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DNA Repair Proteins >
DNA Gyrase (E. coli) |
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Prices are in US dollars and valid only for US orders.
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Description:
DNA Gyrase (E. coli) is a Type II topoisomerase that catalyzes the introduction of negative supercoils in DNA in the presence of ATP. The gyrase holoenzyme is a heterotetramer made up of 2 gyrA (97 kDa) subunits and 2 gyrB (90 kDa) subunits.
Source:
An E. coli strain containing the cloned and expressed gyrA and gyrB genes.
Reagents Supplied:
DNA Gyrase (E. coli) Reaction Buffer
DNA Gyrase (E. coli) Substrate
Properties & Usage
Heat Inactivation:
65°C for 20 minutes
Reaction & Storage Conditions
Reaction Conditions:
1X DNA Gyrase (E. coli) Reaction Buffer
Incubate at
37°C.
1X DNA Gyrase (E. coli) Reaction Buffer:
35 mM Tris-HCl
24 mM KCl
4 mM MgCl2
2 mM DTT
1.75 mM ATP
5 mM spermidine
0.1 mg/ml BSA
6.5 % Glycerol
pH 7.5 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme that catalyzes the conversion of DNA Gyrase (E. coli) Substrate NEB #N0471 to >95% of 0.5 μg of supercoiled plasmid in a total reaction volume of 30 μl in 30 minutes at 37°C.
Concentration:
5,000 units/ml
Unit Assay Substrate: DNA Gyrase (E. coli) Reaction Buffer
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
2 mM DTT
0.1 mM EDTA
50% Glycerol
pH 7.5 @ 37°C
Storage Temperature:
-70°C
Notes
General notes:- The 5X DNA Gyrase Reaction Buffer should be stored at -70°C to maintain optimal stability of components.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of λDNA (HindIII digest)
and 25 units of DNA Gyrase (E. coli) incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 25 units of DNA Gyrase (E. coli) with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.5% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 25 units of DNA Gyrase (E. coli) with 1 μg of
pUC19 DNA for 4 hours at 37ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
References
- Adachi, T. et al. (1987) Nucleic Acids Res., 15, 771-784.
- Higgins, N.P. et al. (1978) PNAS, 75, 1773-1777.
- Swanberg, S.L. and Wang, J.C. (1987) J. Mol. Biol., 197, 729-736.
Reagents Sold Separately
DNA Gyrase (E. coli) Substrate
Companion Products
Topoisomerase I (E. coli)
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