Topoisomerase I (E. coli)

Description

Topoisomerase I (E. coli) catalyzes the relaxation of negatively supercoiled DNA. Topoisomerase I has also been implicated in knotting and unknotting DNA (1) and in linking complementary rings of single-stranded DNA into double-stranded rings (2). The intact holoenzyme is a 97 kDa protein.

Highlights

  • Recognizes non-perfectly matched DNA
  • Isolated from a recombinant source
  • Supplied with 10X Reaction Buffer

Product Source

An E. coli strain containing the cloned topA gene

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
Purified BSA-2010 mg/ml
CutSmart® Buffer-2010X

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme that catalyzes the relaxation of  > 95% of 0.5 μg of pUC19 RF I (negatively supercoiled) DNA in 15 minutes at 37°C in a total reaction volume of 25 μl. DNA supercoiling is assessed by agarose gel electrophoresis in the absence of ethidium bromide.

Reaction Conditions

1X CutSmart® Buffer
Incubate at 37°C

1X CutSmart® Buffer:
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 μg/ml BSA
pH 7.9 @ 25°C

Storage Temperature

-20°C

Storage Conditions

10 mM Tris-HCl
35 mM (NH4)2SO4
50 mM KCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
pH 7.5 @ 25°C

Heat Inactivation

65°C for 20 min

References

  1. Liu, L.F., Depew, R.E. and Wang, J.C. (1976). J. Mol. Biol.. 106, 439-452.
  2. Kirkegaard, K. and Wang, J.C. (1978). Nucl. Acids Res.. 5, 3811-3820.

FAQs

  1. Does Topoisomerase I relax all supercoiled DNA to the same degree or are the products an equilibrium of various conformations?
  2. If the DNA resulting from a Topoisomerase I reaction is run in an ethidium bromide gel and there seems to be no evidence of enzyme activity, what is the most probable explanation?
  3. What is the molecular weight of Topoisomerase I?
  4. Can Topoisomerase I relax both positively and negatively supercoiled DNA?
  5. Does E. coli Toposiomerase I have the same properties as the Vaccinia Topoisomerase I?
  6. Will Topoisomerase I produce single strand-breaks in a plasmid?

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.

Usage Guidelines & Tips

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.