Aug 23, 2024
Pre-Imaging Liquid Growth Medium for Methionine promoted Labeling Systems - 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 Liquid Growth Medium for Methionine promoted Labeling Systems - Yeast. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg329yqv25/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 16, 2024
Last Modified: August 23, 2024
Protocol Integer ID: 99992
Keywords: yeast imaging medium, yeast imaging, yeast medium
Funders Acknowledgement:
NSF
Grant ID: 1917206
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Abstract
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].
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
L-Adenine
Sigma Life Science
Cat#: A-9795
Lot#: 33H12895
L-Methionine
Sigma Life Science
Cat#: M-5308
Lot#: 129H0322
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
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
Compound medium for autoclave
Medium preparation with pre-resolved components8 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 315 mL ddH2O.
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 100 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 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 5 mL Methionine 200x solution.
Note
The additional Methionine represses the Met promoter, which drives PP7 syntheses [2].
Autoclave for 00:15:00 at 121 °C .
Note
Remove the stirring bar before going to autoclave.
When the medium cooled down to around 80 °C add 50 mL sterile Glucose 20%.
Note
The medium can be store at the bench 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
[2] Lari, Azra, et al. "Live-Cell Imaging of mRNP–NPC Interactions in Budding Yeast." Imaging Gene Expression: Methods and Protocols (2019): 131-150.
doi.org/10.1007/978-1-4939-9674-2_9