Maheshri:Media

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General Guidelines

To make 1L of liquid media:

1. In a 2L beaker dissolve everything EXCEPT the sugar (dextrose/galactose/raffinose), amino acids, nucleotides and antibiotics (if any) in ~500 mL water.

2. QS to the appropriate volume (Final volume - volume of sugar and amino acid solutions to be added) with water.

3. Autoclave for 40 min

4. Let cool in the 50°C water bath

5. Use sterile technique to add the sugar, amino acids, nucleotides and antibiotics. Mix.

To make 1L worth of agar plates (~ 40-50 yeast plates, 70-80 bacterial plates):

1. In a 2L beaker dissolve everything EXCEPT the sugar (dextrose/galactose/raffinose), amino acids, nucleotides and antibiotics (if any) in ~500 mL water.

2. QS to the appropriate volume (Final volume - volume of sugar and amino acid solutions to be added) with water.

3. Add 20g of Bacto agar (2% W/V)

4. Autoclave for 40 min

5. Let cool in the 50°C water bath

6. Use sterile technique to add the sugar, amino acids, nucleotides and antibiotics. Mix and pour.

Yeast Media

YePD/YPD

For 1L:

Peptone 20g

Yeast Extract 10g

50% Glucose 40mL

(Plate only) Agar 20g

YPAD,2X

For 1L:

Yeast Extract 20g

Peptone 40g

50% Glucose 80mL

Adenine 200mg

(Plate only) Agar 20g

SD min

For 1L:

Yeast Nitrogen Base w/o Amino Acids 6.7g

50% Glucose 40mL

(Plate only) Agar 20g

SD Dropout

For 1L:

Yeast Nitrogen Base w/o Amino Acids 6.7g

50% Glucose 40mL

10X Amino acid droupout solution 100mL

(Plate only) Agar 20g

5' FOA Plates

For 1L worth of plates:

1. Dissolve 6.7g of Yeast Nitrogen Base w/o Amino Acids, 50mg of uracil, and 20g Agar in 300mL of water. Mix well.

2. QS to 360mL

3. Autoclave for 40 min

4. Let cool in 50°C water bath.

5. Use sterile technique to add the following: 40mL 50% glucose, 100mL 10X AA complete

6. Add 500mL of 5' FOA solution. Mix and pour

W303 Spo Media

For 1L:

10 g potassium acetate (1% final)

1 g yeast extract (0.1% final)

0.5 g dextrose (0.05% final)

If any amino acids are required, add each at the concentration listed under Amino Acid Solution, Complete.

This nitrogen-deficient “starvation” medium contains acetate as a carbon source to promote high levels of respiration, which induces diploid yeast strains to sporulate. Sporulation can be carried out in liquid media or on plates (add 20g agar/L media).

YPA medium (2% potassium acetate, 2% peptone, and 1% yeast extract) is excellent for inducing high levels of respiration in cells prior to sporulation.

Bacterial Media

LB (Luria Broth)

For 1L:

Trypton 10g

Yeast Extract 5g

NaCl 10g

10N NaOH 100μL

(Plate only) Agar 20g

TB (Terrific Broth)

For 1L:

Trypton 10g

Yeast Extract 20g

Glycerol 4mL

(Plate only) Agar 20g

TB Amp plates

To make TB-Amp plates, add 1mL of 1000X ampicillin stock (at 100mg/mL) to 1L of TB media after it has been cooled down to 50°C. Mix and pour.

X-gal

X-gal is located in the -20 freezer as a 20 mg/mL stock in DMSO. It is stored in a light block container. Final concentration for blue-white selection is 40 ug/mL.

Sugar, Amino Acids, Nucleotides and Antibiotics Solutions

50% Glucose

For 1L:

1. In a 2L beaker dissolve 500g of dextrose in 400 mL water

2. Microwave for 5 min

3. Dissolve by mixing and stirring until glucose goes into solution (can take a while)

4. QS to 1L with water

5. Autoclave for 30 min

20% Galactose

For 1L:

1. In a 2L beaker dissolve 200g of galactose in 500 mL water

2. Microwave for 5 min

3. Dissolve by mixing and stirring

4. QS to 1L with water

5. Autoclave for 30 min

40% raffinose

For 1L:

1. In a 2L beaker dissolve 400g of raffinose in 400 mL water

2. Microwave for 5 min

3. Dissolve by mixing and stirring

4. QS to 1L with water

5. Filter sterilize raffinose solution. DO NOT AUTOCLAVE

Amino Acid Solution, Complete

For 1L of 10x stock (good for 10L of media)

Adenine          0.4g
Arginine         0.2g
Histidine        0.2g
Isoleucine       0.3g
Leucine          1.0g
Lysine           0.3g
Methionine       0.2g
Phenylalanine    0.5g
Threonine        2.0g
Tryptophan       0.4g
Tyrosine         0.3g
Uracil           0.2g
Valine           1.5g

1. Dissolve the amino acids with 1L of water in a 2L flask

2. Dissolve by mixing and stirring on a hot plate

3. Aliquot in 100mL bottles

4. Autoclave for 25 min to sterilize

Ampicillin (100mg/mL)

1. Dissolve 1g of ampicillin in 10mL of sterile water (or 70% Ethanol)

2. Filter sterilize

3. Aliquot 1mL

4. Store at -20°C

G418 (200mg/mL)

1. Dissolve 2g of geneticin in 10mL of sterile water

2. Filter sterilize

3. Aliquot 1mL

4. Store at -20°C

Nourseothricin

(for use with Nat gene selection)

1. dissolve 100mg of nourseothricin in 1mL DDH2O (100 mg/mL working stock)

2. filter sterilize

3. store at -20C (only good for 6 months)

Selection concentrations: S. cerevisiae (100 ug/mL), E. coli (50 ug/mL)

5' FOA solution

For 500mL solution (to make 1L 5' FOA plates):

1. Dissolve 1g of 5' FOA in 500mL of water

2. Filter sterilize

3. Warm slightly in 50°C water bath before adding to media with agar to avoid immediate solidification.

Please note that the 5' FOA should be made the same day plates are to be poured.