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Topoisomerases and Enzymatic Activity: A How-To Guide to Establishing Activity

Topoisomerases and Enzymatic Activity: A How-To Guide to Establishing Activity

If you experience low or no activity with our enzymes, consider doing a ‘reconstruction’ type of experiment as follows. Note that this is generally good practice upon receipt of new enzyme from TopoGEN, just to verify that everything is working properly in your lab.
  1. FIRST, When you receive your product, be sure to store as directed by the product literature. Often this means placing immediately at -70ºC. In some cases you can thaw and aliquot the enzyme (not less than 5 ul / tube) to avoid repeated thaw-freeze cycles.
  2. Set up a control experiment designed to test activity in your hands, using your equipment and your lab. The purpose of this simple titration is to establish conditions for separation/resolution of substrate and products. It also demonstrates the marker locations relative to reaction products.

PROCEDURE:

  • Set up three reactions with 0, 0.5 and 1 ul of undiluted enzyme.
  • Incubate for 30 minutes @ 37ºC.
  • Terminate reactions per protocol.
  • Add gel loading dye.
  • Load a 1% gel along with the relevant markers.

For PLASMID BASED ASSAYS:

  • Run a non-ethidium bromide gel (no EB in gel or buffer).
  • Try to run gels NO LONGER than 2 hours (sufficient voltage to get blue dye about 50-75% down the gel).
  • Stain with EB for 10-15 minutes, then destain with water for 10 minutes with a few fluid changes.
  • DO NOT let gels sit around too long before imaging.

For kDNA BASED ASSAYS:

  • Run an EB Gel (0.5ug EB/ml) in gel and buffer.
  • Run the gel at higher voltage to move the dye about 5cm (150v or higher).
  • Run time should be pretty short (less than 30 minutes usually).
  • Destain with water for 10 minutes with a few fluid changes.
  • DO NOT let gels sit around too long before imaging.

Now you can image the destained gels and look for the conversion of supercoiled to relaxed DNA (topo I) or decatenation of kDNA (Topo II). Refer to Fig. 1 below:

Top1_top2_Titration_dataFig 1. Topo I (Left) and Topo II Titration data. Your data should look similar to this. The topo I is plasmid based and topo II is kDNA based. In the case of topo I, the enzyme titration is obvious; in the case of topo II, there was excessive topo II so both lanes (0.5ul and 1 ul) displayed full activity. NOTE: Topo I gel has no EB; topo II gel has EB in gel/buffer.

TopoGEN certifies our enzymes to work with in-house produced DNA substrates (ie DNA substrates from third party sources cannot be guaranteed). Of course, if you still have difficulties with these or other enzymes from TopoGEN, you can always contact us by emailing an image of your experiment to support@topogen.com.  Please be sure to include the lot numbers of the enzyme and substrates used, and the volumes of product you loaded.  We will do everything we can to get you cosmetic results.

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