Psoralen-qPCR supercoiling assay

Warning

This protocol is deprecated. It did not work well with our original UV crosslinker, and could not measure plasmids.

Note

This protocol was used for the experiments described in Babos and Galloway, Cell Stem Cell, 2019 for measuring DNA supercoiling using psoralen intercalation as a metric of negative supercoiling. Psoralen preferentially incorporates into underwound DNA (negatively supercoiled DNA).

Experiment setup

  1. Prepare half of a 6-well plate per replicate. For conditions in triplicate, this means 1.5 6-well plates per condition!

  2. Treat cells according to your experiment.

  3. If you want to arrest the cell cycle prior to measurement, treat the cells with 1 μM aphidicolin for 1.5 to 2 hours prior to cell harvest and DNA extraction.

Chemical

Concentration

Molar mass

mg / mL

Aphidicolin

1 μM

338.48 g/mol