Overview
Making selective yeast plates.
Materials
- Nanopure water
- Yeast nitrogen base (YNB)
- Ammonium sulfate
- Agar
- Sugars (normally 20% dextrose, alternative 20% raffinose/10% sucrose/20% galactose)
- Amino acid dropout solution (in 4°C room)
Procedure
N.B. - this protocol makes ~1 sleeve of moderately thick plates. Adjust volumes as needed.
- Mix:
- 400 mL NP water (or less, if using alternate sugars)
- 0.85g YNB (Stored in cold room)
- 2.5g NH4SO4
- 10g Difco agar (not Bacto agar)
- Mix the solution well, add a stir bar, and autoclave on liquid cycle
- After autoclaving, place flask on stir plate to cool
- When bottle is cool enough to touch with the bare hand, add dropout solution and sugar(s)
- Mix, then pour into plates
- Pour just enough cover the base (without shaking)
- Briefly flame the top of the plate to eliminate bubbles
Notes
- Before autoclaving, the mixture will separate into two layers. It should go into solution after autoclaving.
- If pouring multiple bottles of media, the remaining bottles can stay in the autoclave until needed.
- I've found that it cuts down on condensation if you pile the plates on top of each other while they dry. -Jkm
- If you don't want to flame, drop a stirbar into the flask before autoclaving. Mix on the stirplate to add sugar and aa's, stir some more to take care of bubbles. Pour carefully. -tsb
- Plate thickness varies greatly depending on who pours the plates. I prefer thicker plates (500mL = 24 plates) rather than thinner plates (500mL = 40+ plates). In my experience, thin plates can dry out before the yeast colonies reach their full growth (so you end up with small colonies and thin streaks). This is particularly problematic for slow-growing strains.
Contact
Win
For another protocol, look here.
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