Test crosses

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Test crosses with iodine staining

To test the mating type, simply set up a set of crosses with strains of known mating types.

  1. Take a dollop of cells and make three patches on a mating plate
    • Add to the first patch some cells from a known Plus and mix
    • Add to the last patch some cells from a known Minus and mix
    • Leave the one in the middle alone
  2. You can easily test 21 strains on a single 9cm plate.
  3. Also make a control for the two references you use:
    • grow each alone
    • mix the two together
  4. Incubate for 3 days at 25°C
  5. Stain the cells using iodine and note which cross worked
    • Only the cross with P --> the focal strain was Minus
    • Only the cross with M --> the focal strain was Plus
    • All three --> the focal strain was homothallic
    • None --> the focal is sterile (Better hope you did not forget to add the control cross, because now is a good moment to check if your plate was correct or you used the wrong reference, or something else might have happened).

Alternatively, mating types can be defined by performing PCR for the active mating-type locus.