Aug 23, 2024
Pre-Imaging Solid Growth Medium - Yeast
- Mathias Hammer1,
- Ammeret Rossouw1,
- Azra Lari2,
- Ben Montpetit3,
- David Grunwald1
- 1UMass Chan Medical School, RNA Therapeutics Institute, Worcester, MA, USA;
- 2University of Alberta, Department of Cell Biology, Edmonton, AB, Canada;
- 3University of California, Department of Viticulture and Enology, Davis, CA, USA
Protocol Citation: Mathias Hammer, Ammeret Rossouw, Azra Lari, Ben Montpetit, David Grunwald 2024. Pre-Imaging Solid Growth Medium - Yeast. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5r9wjg1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 13, 2024
Last Modified: August 23, 2024
Protocol Integer ID: 99629
Keywords: yeast solid medium
Funders Acknowledgement:
NSF
Grant ID: 1917206
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Abstract
This protocol describes the steps to prepare solid culture medium for Saccharomyces cerevisiae.
This solid medium is used for cultures that are going to be investigated by fluorescence imaging. Of that reason the medium is prepared with an abundance of Adenine to reduce auto-fluorescence [1].
Materials
SC-Ura Powder
Sunrise Science Products
Cat#: 1306-030
Lot#: 23K3083
Exp: 10/2027
Yeast Nitrogen Base Without Amino Acids
Sigma Life Science
Cat#: Y0626-250G
Lot#: SLBG0555V
Glucose
Sunrise Science Products
Cat#: 1907-1kg
Lot#: 3A0036
Agar
Sunrise Science Products
Cat#: 1910-500
Lot#: 3B0104
Sodium Hydroxide
Fisher Scientific
Cat#: S318-500
Lot#: 130802
L-Adenine
Sigma Life Science
Cat#: A-9795
Lot#: 33H12895
Deionized Water
Equipment:
500 ml laboratory bottle with screw cap
1ml pipette
50 ml pipette
stirring hot plate
magnetic stirring bar
micro scales
autoclave
thermometer
10cm polystyrene Petri dishes (20 pieces)
4°C fridge
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)
Sodium Hydroxide 1M solution 50 ml:
Concentration: 39.997 g/l
mix 1.99985 g NaOH into 50 ml ddH2O
Adenine 100x 100 ml solution:
Concentration 3 g/l
mix 0.3 g Adenine in 100 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
Compound medium for autoclave
2m
Medium preparation with pre-resolved components13 steps
This version of the protocol shows the preparation of the medium from SC-XX 10x and YNB 20x solutions.
Fill a 500 ml flask with 369 mL 2H2O.
Add a magnetic stirring bar and place the flask on a stirring hot plate.
Add 25 mL YNB 20x solution (Yeast Nitrogen Base with Ammonium Sulfate without Amino Acids).
Add 50 mL SC-XX 10x solution.
Note
In regard to cover all optional dropout media the amino acid base holds the notification -xx, where xx stand for the amino acid(s) that is as selection factor, missing in the medium.
Add 10 g Agar
Add 5 mL Adenine 100x solution
Note
The additional Adenine is supposed to repress the Adenine synthesize to reduce a possible accumulation of red pigment [1].
Add 1 mL NaOH 1 molar solution.
Note
This is essential for the solidification of the medium!
Autoclave for 00:15:00 at 121 °C .
Note
Remove the stirring bar before going to autoclave.
Cooling and platting
Add a sterile stirring bar and thermometer. Let the medium stir to homogenize its temperature, while cooling.
When the medium cooled down to around 80 °C add 50 mL sterile Glucose 20%.
When the medium cooled down to 60 °C , plate 25 mL per 10 cm Petri dish (~20 dishes).
Let the medium solidify and cool down to Room temperature .
Seal the prepared dishes in a plastic bag to prevent condensation and store them in 4 °C fridge.
Note
The agar plates can be store in the 4°C fridge for 2 to 3 months.
Protocol references
[1] Kokina, Agnese et al. "Adenine auxotrophy–be aware: some effects of adenine auxotrophy in Saccharomyces cerevisiae strain W303-1A." FEMS yeast research 14.5 (2014): 697-707.
doi:10.1111/1567-1364.12154