Pichia Adenine Dropout (PAD) agar

(1) Prepare SMM base

Synthetic minimal medium (SMM) 2 x base

Component Concentration Quantity per L

Yeast Nitrogen Base (without amino acids or (NH4)2SO4) 0.34% 3.4 g

Dextrose 2% 20 g

(NH4)2SO4 1% 10g

H2O - 500 ml

(2) Add the following stock solutions (prepared in advance) to the 2x SMM base medium. Stock
solutions should be autoclaved and stored at 4 C unless otherwise specified.

Component Stock concentration Volume stock per litre Final concentration in

(g/100 mL) of medium (mL) medium (mg/L)
a
Uracil 0.2 10 20
L-Tryptophan 1 2 20
L-Histidine HCl 1 2 20
L-Arginine HCl 1 2 20
L-Methionine 1 2 20
L-Tyrosine 0.2 15 30
L-Leucine 1 10 30
L-Isoleucine 1 3 30
L-Lysine HCl 1 3 30
L-Phenylalanine a 1 5 50
a
L-Glutamic acid 1 10 100
L-Aspartic acid a,b 1 10 100
L-Valine 3 5 150
L-Threonine a,b 4 5 200
L-Serine 8 5 400
a
Store stock at room temperature to prevent precipitation
b
Filter sterilize and add after autoclaving medium and cooling to ~50 C

(3) Add 20 g Bacto agar per litre of complete medium.

(4) Adjust volume to 1 L with distilled water.
(5) Autoclave on liquid cycle (15 minutes at 121 C)
(6) Cool to 50 C, add aspartic acid and threonine stock solutions and mix.
(7) Pour agar plates (1 L should make about 30 – 35 plates)