Growing a Liquid Yeast Culture

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Tony Rook
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Growing a Liquid Yeast Culture

Please find the following protocol:

Growing a Liquid Yeast Culture

Overview:

This is a general protocol for growing liquid yeast cultures.

Procedure:

1. Flame the opening of a sterile flask and the mouth of the sterile media bottle.

2. Immediately, using sterile technique, pipet or pour YPD or Minimal Media into the flask. Do not fill more than two-thirds full so that there is plenty of room for aeration of the culture.

Note: You will want to grow some strains under selective pressure to, for example, select for a plasmid or a gene knock-out. In that case, inoculate Minimal Media with the strain of interest. The Minimal Media needs to be supplemented with the amino acids that the strain requires for growth. To select for plasmids containing the URA3 gene, cells can be grown in Minimal Media -Ura or in Cas Amino Acids Media.

3. Pick a single yeast colony from a plate with a sterile stick. Make sure not to pick small, whitish petites. Petites have mitochondrial defects that prevent oxidative metabolism.

4. Transfer the colony to the liquid media in the flask. Do not tighten cap completely.

5. Incubate the flask at 30°C with moderate shaking. If the strain is temperature sensitive, grow the strain at 23°C or 25°C. To grow cells at the restrictive temperature, place the culture at 37°C.

Solutions:

Uracil Supplement (100X).........................200 mg/100 ml Uracil
.........................................Autoclave
Arginine Supplement (250X)..............1 g/100 ml L-Arginine-HCl ......................................Autoclave
Serine Supplement (200X)..........................8 g/100 ml L-Serine .........................................Autoclave
Threonine Supplement (200X)................4 g/100 ml L-Threonine
....................................Autoclave
Valine Supplement (200X)...........................3 g/100 ml L-Valine
.........................................Autoclave
Glutamic Acid Supplement (100X).....1 g/100 ml L-Glutamic Acid ..............................Autoclave
Adenine Supplement (100X).......200 mg/100 ml Adenine Sulfate
......................................Autoclave
Cas Amino Acids Media....Add appropr. Amino Acid Supplements
.............................................Autoclave
...............6.7 g/liter Yeast Nitrogen Base
..................................20 g/liter Glucose
Minimal Media..6.7 g/liter Yeast Nitrogen Base (w/o amino acids)
.............Add appropriate Amino Acid Supplements
..........................................................Autoclave
...............................................20 g/liter Glucose
YPD...........Add 20% (w/v) sterile Glucose to a final conc. of 2%
..................................Autoclave and allow solution to cool.
....................................................20 g/liter Bacto Peptone
............................................10 g/liter Bacto Yeast Extract
Aspartic Acid Supplement (100X)....1 mg/100 ml L-Aspartic Acid ...............................Autoclave
Isoleucine Supplement (150X)...............1 g/100 ml L-Isoleucine ...................................Autoclave
Phenylalanine Supplement (200X).....1 g/100 ml L-Phenylalanine ..............................Autoclave

Tyrosine Supplement (40X)...............200 mg/100 ml L-Tyrosine ..................................Filter Sterilize
.....................Store covered with foil
Lysine Supplement (200X)....................1 g/100 ml L-Lysine-HCl .........................................Autoclave
Leucine Supplement (150X).......................1 g/100 ml L-Leucine
.......................................Autoclave
Methionine Supplement (200X).......400 mg/100 ml L-Methionine
...................................Autoclave
Histidine Supplement (200X)........400 mg/100 ml L-Histidine-HCl .....................................Autoclave
Trptophan Supplement (200X).......400 mg/100 ml L-Tryptophan ...................................Autoclave

Bioreagents and Chemicals:

Bacto-Peptone
L-Valine
Bacto Yeast Extract
L-Tyrosine
Uracil
L-Asparic Acid
L-Arginine HCl
Adenine Sulfate
L-Tryptophan
L-Threonine
Yeast Nitrogen Base, Amino Acid Free
L-Serine
Glucose
L-Phenylalanine
L-Methionine
L-Histidine HCl
L-Lysine HCl
L-Leucine
L-Isoleucine
L-Glutamic Acid

Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p1992