Answer to Question #92812 in Molecular Biology for Naad

Question

You are studying a protein that you suspect has DNA topoisomerase activity. Describe how you would test the protein for this activity in vitro.

Answer 1

The reagents to be used include distilled water, loading dye, proteinase K, agarose gel, and ethidium bromide.

Method

1.      Mix the distilled water, 10x assay buffer, supercoiled DNA, and the test extract.

2.      Incubate the mixture for 30 minutes at 37 degrees Celsius

3.      Add 5 micro litres stop loading dye

4.      Add proteinase and digest for 30 minutes

5.      Load 1% agarose gel and use 1x TAE buffer

6.      Run the gel

7.      Stain with ethidium bromide

8.      Destain using distilled water for 30 minutes

9.      Photograph using UV transilluminator