Takara DNA Topoisomerase I

More Information

Applications

• Conversion and analysis of DNA conformation

Buffer components

• 10X DNA Topoisomerase I Buffer
  350 mM Tris-HCl (pH 8.0), 720 mM KCl, 50 mM MgCl2, 50 mM DTT, and 50 mM spermidine
• 0.1% BSA

Source

Calf thymus

Storage

–20°C

Unit definition

One unit is the amount of the enzyme that completely relaxes 0.5 µg of supercoiled pBR322 DNA in 50 µl of the reaction mixture in 30 min at 37°C.

Concentration

20 units/µl

Notes

• The supplied reaction buffer is added with spermidine at final concentration of 5 mM. Without adding spermidine, the enzyme activity may decrease to 1/20.

• When supercoiled molecules such as pBR322 DNA or phiX174 DNA RF I are treated with DNA Topoisomerase I and are analyzed with electrophoresis on an agarose gel that contains EtBr, no change in the position of the bands occurs with the enzyme treatment. This is because DNA, which is converted to the relaxed form by DNA Topoisomerase I, resumes its supercoiled configuration when treated with EtBr. Thus, when the activity of DNA Topoisomerase I is to be measured, we recommend that electrophoresis be done with an agarose gel that does not contain EtBr, and then staining with EtBr be done afterwards.

• White precipitate may be generated when adding 0.1% BSA directly to the supplied buffer. Prepare the reaction mixture (final conc. 0.01% BSA) by adding the reagents in the order given below.

  dH2O → supplied buffer → 0.1% BSA → substrate DNA

• This enzyme easily sticks to glass or plastic material. BSA should be added to the reaction mixture.

Additional product information

Please see the product`s Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.

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