DNA Gyrase (E. coli)

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.

Product Source

An E. coli strain containing the cloned gyrA and gyrB genes.

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
DNA Gyrase (E. coli) Reaction Buffer5X

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme that catalyzes the conversion of 0.5 µg DNA Gyrase (E. coli ) Substrate to >95% supercoiled plasmid in a total reaction volume of 30 µl in 30 minutes at 37°C.

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

Storage Temperature

-80°C

Storage Conditions

10 mM Tris-HCl
50 mM KCl
2 mM DTT
50% Glycerol
0.1 mM EDTA
pH 7.5 @ 37°C

Heat Inactivation

65°C for 20 min

Supplied As

Liquid

Notes

  1. The 5X DNA Gyrase Reaction Buffer should be stored at -80°C to maintain optimal stability of components.

References

  1. Adachi, T. et al. (1987). Nucleic Acids Res. 15, 771-784.
  2. Higgins, N.P. et al. (1978). PNAS. 75, 1773-1777.
  3. Swanberg, S.L. and Wang, J.C. (1987). J. Mol. Biol. 197, 729-736.

FAQs

  1. Is the linking number (ΔLk) or the superhelical density (σ) that results from the gyrase reaction fixed or random? Does the sequence, GC content or size of the circular DNA affect the linking number?
  2. Will E. coli DNA Gyrase add additional negative supercoils in already supercoiled DNA?
  3. Do you offer a reverse gyrase?
  4. How should a DNA treated with gyrase be run on a gel?
  5. Can E. coli DNA Gyrase supercoil close and open circular DNA?

Protocols

  1. Standard E. coli DNA Gyrase Reaction (M0306)

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Interactive Tools

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Endonuclease Activity (Nicking):
    The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
  • Exonuclease Activity (Radioactivity Release):
    The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
  • Non-Specific DNase Activity (16 hour):
    The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
  • Non-Specific RNase Activity (16 hour, RNA):
    The RNA is tested in a reaction under standard reaction conditions. After incubation for 16 hours there is no detectable degradation of the RNA as determined by agarose gel electrophoresis.

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.