DNA Topoisomerase I (Vaccinia Virus), 1000 U (for Nuclease/Topoisomerase)

Product Information

Product Description

DNA topoisomerase I produced by our company is a type I eukaryotic topoisomerase derived from vaccinia virus . This DNA Topoisomerase I has DNA Relaxing activity, which can make double-stranded covalently closed circular positive or negative supercoiled DNA unsupercoiled, thereby forming double-stranded circular DNA molecules with less positive or negative supercoil. Vaccinia DNA Topoisomerase I can also make double-stranded DNA form knots (knotting) or unknots (unknotting), so that complementary single-stranded circular DNA becomes double-stranded circular DNA.

In addition, Vaccinia DNA Topoisomerase I can specifically recognize the 5'- (C/T)CCTT-3' sequence in double-stranded DNA, and can open the phosphodiester bond after the 5'- (C/T)CCTT sequence on one strand. The energy released by breaking the phosphodiester bond can catalyze the 3' phosphate group at the DNA incision to form a covalent ester bond with the 274th tyrosine (Try) hydroxyl group of the topoisomerase, thus forming a covalent complex between DNA and the enzyme. At the same time, the covalent ester bond formed can be attacked by the 5' hydroxyl group of the cut single-stranded DNA, and the previous phosphodiester bond can be restored without the need for ATP and DNA ligase, that is, the DNA is reconnected and Vaccinia DNA Topoisomerase I is released at the same time.

Currently, DNA Topoisomerase I (vaccinia virus) is often used as a new tool enzyme for DNA recombination and ligation, and is used for ligation of DNA vectors and recombinant fragments, nick repair, and linker ligation.

Features:  Unwinds DNA supercoils and specifically opens phosphodiester bonds.

Source: E. coli strain carrying Vaccinia virus topoisomerase gene; its molecular weight is about 37KDa;

Enzyme activity definition: The amount of enzyme required to completely unwind 1 µg of pUC19 vector by incubation at 37°C for 30 min is defined as one unit of enzyme activity.

Purity and concentration: Purity detected by SDS-PAGE 95%; residual endogenous nucleic acid <1 pg/μL (qPCR detection); 10 U/μL.

Inactivation or inhibition: Heating at 80°C for 20 minutes can fully inactivate DNA Topoisomerase I.

Enzyme storage buffer: 50 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 0.1 M NaCl, 0.1% Triton X-100, 50% glycerol, pH 7.5.

10х Reaction buffer: 500 mM Tris-acetate, 1 M NaCl, 25 mM MgCl ₂ , 1 mM EDTA, pH 7.5.

Storage and transportation

Low temperature transportation; storage at -20, valid for one year;

composition

Reference

1. Reference reaction system:

2. After setting up the system as above, mix gently and then centrifuge the liquid to the bottom of the tube.

3. Incubate at 37°C for 30 minutes and place on ice to terminate the reaction.

4. When performing agarose gel electrophoresis to detect the reaction products, it is necessary to use agarose gel that does not contain ethidium bromide (EB). After the electrophoresis, nucleic acid staining and gel imaging observation should be performed.

Precautions

1. Because when the gel contains ethidium bromide, it will bind to the bases of DNA and catalyze the formation of supercoils. Therefore, when detecting the plasmid product treated with DNA Topoisomerase I (vaccinia virus) by agarose gel electrophoresis, gel without ethidium bromide (EB) should be used for electrophoresis. After the electrophoresis is completed, ethidium bromide or other suitable nucleic acid dyes should be used for nucleic acid staining detection of the gel.

2. When using this product, the enzyme should be stored in an ice box or on an ice bath. After use, it should be stored at -20ºC immediately.

3. For your safety and health, please wear a lab coat and disposable gloves when operating.

This product is for scientific research purposes only and not for clinical diagnosis!

https://www.servicebio.com/goodsdetail?id=12045