TITLE: Pre-Imaging Liquid Growth Medium for Methionine promoted Labeling Systems - Yeast
AUTHORS: Mathias Hammer, Ammeret Rossouw, Azra Lari, Ben Montpetit, David Grunwald

[DESCRIPTION]
This protocol describes the steps to prepare liquid culture medium for Saccharomyces cerevisiae. This liquid medium is used to optimize yeast cultures for fluorescence imaging by the reduction of auto-fluorescence through an abundance of Adenine [1].
The surplus of Methionine in the medium represses the pp7-CP expression, which reduces the level of CP aggregates [2].

[BEFORE_START]
Have the following solutions premixed:

Glucose 20% 500 ml solution:
Concentration: 200 g/l
mix 100 g Glucose in 500 ml deionized water (ddH2O)

Adenine 100x 100 ml solution:
Concentration 3 g/l
mix 0.3 g Adenine in 100 ml ddH2O

Methionine 200x 50 ml:
Concentration: 17.12 g/l
mix 856 mg into 50 ml ddH2O

Optional:
SC-xx 10x 100ml solution:
Concentration: 19.2 g/l
mix 1.92 g into 100 ml ddH2O

YNB 20x 100ml solution:
Concentration: 134.4 g/l
mix 13.44 g into 100 ml ddH2O

[STEPS]
SECTION: Preparation of 500ml liquid pre-imaging growth medium for Methionine promoted Labeling Systems
1. Compound medium for autoclave
SECTION: Preparation of 500ml liquid pre-imaging growth medium for Methionine promoted labeling systems
1.1. Fill a 500 ml flask with 440 mLddH2O. 
Add a magnetic stirring bar and place the flask on a stirring hot plate.
SECTION: Preparation of 500ml liquid pre-imaging growth medium for Methionine promoted labeling systems
1.2. Add 3.36 g Yeast Nitrogen Base with Ammonium Sulfate without Amino Acids (YNB).
SECTION: Preparation of 500ml liquid pre-imaging growth medium for Methionine promoted labeling systems
1.3. Add 0.86 g SC-XX powder. 
 

SECTION: Preparation of 500ml liquid pre-imaging growth medium for Methionine promoted labeling systems
1.4. Add 5 mLAdenine 100x solution.
 

SECTION: Preparation of 500ml liquid pre-imaging growth medium for Methionine promoted labeling systems
1.5. Add 5 mLMethionine 200x solution.
 

SECTION: Preparation of 500ml liquid pre-imaging growth medium for Methionine promoted labeling systems
2. Autoclave for 15 min at 121 °C.
 

SECTION: Preparation of 500ml liquid pre-imaging growth medium for Methionine promoted labeling systems
3. When the medium cooled down to around 80 °C add 50 mL sterile Glucose 20%.
SECTION: Preparation of 500ml liquid pre-imaging growth medium for Methionine promoted labeling systems
4. 
 

SECTION: Preparation of 500ml liquid pre-imaging growth medium for Methionine promoted Labeling Systems
1.1. Fill a 500 ml flask with 315 mLddH2O. 
Add a magnetic stirring bar and place the flask on a stirring hot plate.
SECTION: Preparation of 500ml liquid pre-imaging growth medium for Methionine promoted Labeling Systems
1.2. Add 25 mLYNB 20x solution (Yeast Nitrogen Base with Ammonium Sulfate without Amino Acids).
SECTION: Preparation of 500ml liquid pre-imaging growth medium for Methionine promoted Labeling Systems
1.3. Add 100 mL SC-XX 10x solution.
 

SECTION: Preparation of 500ml liquid pre-imaging growth medium for Methionine promoted Labeling Systems
1.4. Add 5 mLAdenine 100x solution.
 

SECTION: Preparation of 500ml liquid pre-imaging growth medium for Methionine promoted Labeling Systems
1.5. Add 5 mLMethionine 200x solution.
 

SECTION: Preparation of 500ml liquid pre-imaging growth medium for Methionine promoted Labeling Systems
2. Autoclave for 15 min at 121 °C.
 

SECTION: Preparation of 500ml liquid pre-imaging growth medium for Methionine promoted Labeling Systems
3. When the medium cooled down to around 80 °C add 50 mL sterile Glucose 20%.
SECTION: Preparation of 500ml liquid pre-imaging growth medium for Methionine promoted Labeling Systems
4.