Maheshri:Media
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 and 50mg of uracil in 300mL of water. Mix well.
2. QS to 360mL
3. Autoclave for 40 min
4. Let cool
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.
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
5 FOA
For 1L:
1. Dissolve 2g of 5FOA in 1L of water
2. Filter sterilize
Please note that the 5 FOA should be made the same day plates are to be poured
