Manual

Product Number: DB004

Shipping and Storage

Low temperature transportation; Shelf life of one year at -20℃.

Components

Description

The DNA topoisomerase I produced by our company is a type I eukaryotic topoisomerase derived from the smallpox virus. The DNA Topoisomerase I has DNA Relaxing activity, which can cause double stranded covalently closed circular positive or negative supercoiled DNA to undergo supercoiling, thereby forming double stranded circular DNA molecules with fewer positive or negative supercoils. Vaccinia DNA Topoisomerase I can also cause double stranded DNA to form knots or undo knots, resulting in complementary single stranded circular DNA becoming 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 a phosphodiester bond on one strand after the 5 ‘-… (C/T) CCTT↓sequence. 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 topoisomerase, thus forming a covalent complex between DNA and the enzyme. The covalent ester bond formed simultaneously can be attacked by the 5 ‘hydroxyl group of the cleaved single stranded DNA, and without the need for ATP and DNA ligase, the previously formed phosphodiester bond can be restored, that is, the DNA can be reconnected while releasing Vaccinia DNA Topoisomerase I.

Application

At present, DNA Topoisomerase I (vaccine virus) is often used as a novel tool enzyme for DNA recombination and ligation, for the ligation, defect repair, and linker ligation of DNA vectors and recombinant fragments.

Features

1. Features: DNA supercoiling and specific opening of phosphodiester bonds.
2. Source: Escherichia coli strains carrying the Vaccinia virus topoisomerase gene; Its molecular weight is about 37KDa;
3. Enzyme activity definition: The amount of enzyme required to completely unwind 1µg of pUC19 carrier after incubation at 37℃ for 30 minutes is defined as one enzyme activity unit.
4. Purity and concentration: SDS-PAGE detection shows a purity of ≥95%; Endogenous nucleic acid residue<1pg/μL (qPCR detection); 10U/μL。
5. Inactivation or inhibition: Heating at 80 ° C for 20 minutes can fully inactivate DNA Topoisomerase I.
6. Enzyme storage buffer：50mM Tris-HCl，1mM DTT，0.1mM EDTA，0.1 M NaCl，0.1%Triton X-100，50%glycerol，pH 7.5.
7. 10х Reaction buffer：500mM Tris-acetate, 1M NaCl, 25 mM MgCl_2, 1mM EDTA，pH 7.5.