Halo Assay

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Reagents

  • Fresh over night cultures of yeast strains
  • YPD medium
  • 1% sterile agar
  • YPD plates, warmed to ~42°C

Stock solution of the relevant drug Sterile paper filter disks (Whatman 3MM paper disks cut out with a perforator, autoclaved and dried)

Method

  1. Prepare a dilution series (4 dilutions) of your antibiotic
  2. Dilute 40 µl of the stationary culture into 2 ml of fresh YPD medium.
  3. Add 2 ml of molten agar to the tubes, mix quickly and poor over the surface of a pre-warmed agar plate. Spread as evenly as possible before the agar sets.
  4. Per plate to be prepared, place four paper filter discs onto clean parafilm. Spot 48 µl of each dilution onto one of the disks, and place the disk onto the set agar surface of the YPD plate.
  5. Incubate at 25º (or other relevant growth temperature) until halos are clearly visible. Scan the plates and determine the diameter of the halos using ImageJ.
  6. Plot the halo diameter against the log10 of the drug concentration. You should obtain a straight line. The offset of the lines between wild type and mutant strains is quantitatively related to the change in sensitivity between the strains.

Stock Solutions

Paromomycin: 500 mg/ml, 4 doubling dilutions

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