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Topoisomerase I (vaccinia)

No Product Name Cat.# Conc Size Price(₩) Add
1 Topoisomerase I (vaccinia) M017S 10 units /μl 1,000 units ₩ 200,000 추가
2 Topoisomerase I (vaccinia) M017L 10 units /μl 5,000 units ₩ 550,000 추가
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Product description

Topoisomerase I (vaccinia) catalyzes the relaxation supercoiled DNA molecules by breakage and relegation of phosphodiester bond in a single strand of DNA. This results in the removal of supercoils (either positive or negative superhelical turns). Topo I does not require Mg2+ to function, but low concentrations may increase its activity. Topo I from vaccinia virus is a type I eukaryotic topoisomerase that cleaves DNA 3’ to the target sequence [5’ (C/T)CCTT]. The other DNA strand is not cleaved. Subsequent religation of the phosphodiester bond results in DNA with fewer superhelical turns.

If the (C/T)CCTT site is at the end of the DNA molecule, those bases 3’ of the nick dissociate from the Topo I-DNA complex. Topo I may then create a recombinant molecule by joining the cleaved (C/T)CCTT-containing DNA with another DNA duplex if the other DNA duplex can basepair with the noncleaved strand. Topo I can also form recombinant molecules with the 5’-end of RNA molecules.

            

Applications

- Increase restriction endonuclease digestion of resistant DNA substrates by unwinding the DNA coils to expose

  restriction sites.

- Study vital processes including replication, transcription, and recombination.

- Study DNA structure and topology such as: the effects of supercoiling on transcription in vitro, chromatin

  reconstitution in vitro, and the degree of supercoiling of DNA.

- Assay mutant plasmids which differ in length by only one base-pair.

 

Quality control

- Endonuclease-free

- Exonuclease-free

            

Unit Definition

One unit is defined as the amount of enzyme that converts 1 μg of supercoiled closed circular (Form I) pUC19 DNA to relaxed closed circular form (Form II) in 1 hr at pH 7.5 at 37. DNA supercoiling is assessed by agarose gel electrophoresis in the absence of ethidium bromide.

 

Reaction Conditions

10X Topoisomerase I buffer

Incubate at 37

 

10X Topoisomerase I (vaccinia) buffer

500 mM Potassium Acetate, 200 mM Tris-acetate, 100 mM Magnesium Acetate, 10 mM DTT, pH 7.9 @ 25.

 

Storage

10 mM Tris-HCl, 35 mM (NH4)2SO4, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol pH 7.5 @ 25, Store at -20.

 

Heat Inactivation

65 for 20 min

 

Standard reaction conditions         

Topoisomerase I (vaccinia) Protocol

Plasmid DNA

10 μg

10X Topoisomerase I buffer

5 μl

Topoisomerase I, vaccinia (10 units/μl)

1 μl

Distilled water

up to 50 μl

→ Incubate the reaction mixture at 37. 

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