Single cell colony
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We make single cell colonies for many reasons and the procedure in the end is always the same, only the starting material and the plates differ for each purpose.
uses of single cell colonies
- Random spore analysis (on YES after glusulase treatment)
- Mating-type switching stability (on PMG)
- After electroporation or chemical transformations (don't make dilutions)
If plating out Make sure the medium
Plate out liquid
- Take your cells
- Dilute them into the appropriate medium
- Dilute your cells to 1000 to 10000 cells/ml
- Pipette 50 to 200 ul of the dilution. Use enough liquid to spread well and not too much, because then the spores swim around and you don't get a good spread. Depending on the purpose, you want to aim for about 200-1000 colonies/ plate. When scoring for homothallism/heterothallism you can go up to 10000 cells per plate for iodine staining
- Spread your cells evenly on the plate using a sterile spatula or sterile glass beads
Random spore analysis
- Take sporulated cells from a mating plate with a toothpick
- Add cells to 500 μl of sterile MQ and add 10μl glusulase (aliquote in fridge 1)
- Incubate at 32°C for 16h.
- Check if no asci remain
- Dilute if needed (probably this is needed)
- Spread out as described above (~200 to 500 spores per plate). Use rich medium, or the spores won't germinate (G418 can immediately be added if desired).
- Keep the spore-glusulase mix in the fridge, where it remains fine for about a week.
- Germinate at 32°C and use replicate plating to screen for markers.