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HARRY SCHERTHAN and EDGAR TRELLES-STICKEN
- RNaseA working solution: Use RNaseA at 200 Ilglml in 2xSSC
- Rubber cement (Fixogum; Marabu). Bicycle repair fluid may serve as a
substitute.
- Sodium N-lauroylsarcosine (Fluka Chemie), 2% in dHzO, store at 4°e.
- Solution I (Sol I): 1 M Sorbitol, pH 7. May be stored at 4°C up to
2 weeks.
- Solution II (Sol 11): 0.1 M MES [2-(N-morpholino)-ethane sulfonic
acid]; 1 mM EDTA; 0.5 mM MgClz; 1 M sorbitol; adjust pH to 6.4
with NaOH.
- Sporulation medium: 2% Potassium acetate, pH 7.0. Make up fresh for
each use.
- SSC, 20x: 3 M NaCl, 0.3 M sodium citrate, pH 7.0
- Sucrose
- Target unmasking fluid (TUP; Dianova)
- Tween-20 (Sigma)
- Yeast extract (Difco)
- YPD agar: YPD + 2% Bacto agar
- YPD medium: 1% (w/v) Yeast extract; 2% Bacto peptone; 2% D-glucose
- Zymolyase lOOT (Kirin Brewery Ltd.) Make up a stock solution (10 mg
Zymolyase 100T/mi dHzO) and store at -70°e.
Subprotocol 1
Cell culture
Procedure
1.1 Culture of vegetative yeast cells
1. Streak out yeast strain of interest on a freshly prepared YPD-plate and
incubate for 2 days at 30°e.