Random spores isolation

Resuspend the sporulated cells in 50μl of sterile TBS or PBS (« buffer »).
Prepare serial dilutions :
Tube 1 : 1000μl of buffer
Tube 2 : 100μl of buffer
Tube 3 : 100μl of buffer
Tube 4 : 100μl of buffer

FUME HOOD & Gloves: Add 150μl of diethylether (TOXIC & HIGHLY FLAMABLE) to the cells (precise amount is not important, bt the cells must be imerged).
Vortex hard for 3 minutes.

Pipet 10μl of treated cells into Tube1, Vortex.
Pipet 10 μl of Tube 1 into Tube2. Pipet 2 μl of Tube 1 into Tube 3. Pipet 10 μl of Tube 2 into Tube4. Plate 100 μl of Tubes 1 to 4 onto separate YPD or selective plates.


Tetrad dissection

Dissection plates:
YPD plates + 1M Sorbitol. Autoclave at 1bar, stir well after autoclaving and pour as soon as cooled down to 55°C. DO NOT MAKE THICK PLATES.

Zymolyase solution:
0.5mg/ml ( = 10U/ml) zymolyase (ref 120491 from SEIKAGAKU distributed by MPbiomedicals) in 1M sorbitol. Best if prepared fresh but can be stored in single-use 50 μl aliquots at -20°C for convenience.

Spore preparation:
Dry a dissection plate in incubator for 30min
Resuspend spores into 50μl of zymolyase solution. Incubate 5-10 minutes at 30°C. Put on ice.
Spread some of the spore suspension on the border (up to 2.5 cm from the edge) of a dissection plate.

Follow carefully instructions relative to dissecting microscope (Nathalie Grandin).

Dernière mise à jour : ( 18-12-2007 )