For Bio 284

• 0.15 g YNB
• 0.52 g ammonium sulfate
• 2.0 g agar
• 82 mL H2O
• 8.0 mL 1 mg/mL adenine
• 10.0 mL 20% glucose (final conc = 2g/100 mL)

Minimal Vitamin Medium plus Adenine

Label plates with "+ ADE"

Make sure it's a plus sign!

Autoclave an appropriately sized graduated cylinder to measure the glucose and adenine solutions.

Mark with two blue lines

*Yeast Nitrogen Base without amino acids and ammonium sulfate

Minimal Vitamin Medium for Bio 284

Autoclave an appropriately sized graduated cylinder to measure the glucose solution.

*Difco Yeast Nitrogen Base w/o Amino Acids and w/o Ammonium Sulfate

Mark with a single blue line

Plates for Jeff Laney's Cell & Molecular Biology Lab

SC-Leu low-Ade plates for Jeff Laney's cell and molecular biology lab

SC minus (adenine, histidine, leucine, tryptophan)

Sunrise Science 1330-030

4X will dissolve. Almost completely clear after overnight stirring. Crystal clear after autoclaving.

From the manufacturer:

Commonly added to YNB, nitrogen and glucose, at suggested g/L, for a complete yeast media. Mix with stirring for 10-15 minutes, and filter sterilize or autoclave at 121°C for 15 minutes. To prepare plates, autoclave agar separately and add to sterile medium, or add agar to liquid medium and adjust to pH 5.8-6.0 before autoclaving.

Suggested g/L: 1.64

Storage Temperature: 2-8 C

Jeff's recipe: 10X SC –AHLT 16.4 g/L

Dissolve 16.4 g of Sunrise Science SC –Adenine, –Histidine, –Leucine and –Tryptophan in 1 L distilled water and filter sterilize.

Store in the dark at 4 ˚C.

Will not go into solution.

Concentrate to make SC-L High Ade or Low Ade agar

Make this concentrate, then add adenine hemi-sulfate.

for low-Ade, add 5 mg Ade per L of concentrate

for high-Ade, add 1.6 g Ade per L of concentrate

to make high-Ade from low-Ade concentrate, add 1.595g Ade per L, or 0.1595g per 100 mL

Then filter sterilize. For every L of medium, combine 20g agar autoclaved in 875 mL water with 125 mL warmed up concentrate.

add 125 mL concentrate to autoclaved 875 mL water + 20 g agar

Liquid Medium

Using 4X SC-AHLT (because 10x won't go into solution):

Bring to final volume with water, then filter sterilize.

Jeff Laney's original (discontinued - because you can't make 10X SC-AHLT) recipe:

Filter sterilize

Store at room temperature

Yeast Extract Adenine Dextrose Medium

Ade mutants grow well on this without turning red. Better for keeping the parental strains white longer.

1 gram Yeast Extract 2 grams anhydrous dextrose (glucose) 2 grams Agar 8 mL adenine stock solution (1 mg/mL)

Adenine Stock Solution

400 mg adenine in 400 ml water (1 mg/ml) Store at room temperature. Use 2 ml stock solution for 100 ml of medium; reduce the water added to the medium by 2 ml.

Remember to autoclave an appropriate sized graduated cylinder to measure the glucose solution in the sterile hood.

Yeast Extract Dextrose

For Bio 284

Remember to autoclave an appropriate sized graduated cylinder to measure the glucose solution in the sterile hood.

Mark with a single red line.

Sporulation Medium for Bio 284

OR

• 20.4 mL 5M KOAc per liter
• 12.75 mL 8M KOAc per liter Fisher AAJ63372AE

Mark with a single black line.

YEPAD medium (Yeast Extract + Peptone + Adenine + Dextrose) contains the same ingredients as YEAD but has peptone added. This is a very rich medium used for storing yeast strains.

1 gram Yeast Extract 2 grams Peptone 2 grams anhydrous dextrose (glucose) 2 grams Agar (agar-agar; gum agar) 8 mL adenine stock solution 92 ml water

A Dohlman Lab Protocol

Yeast strains, transformed or untransformed, can be maintained as colonies on solid media at 4° and restreaking every 2 to 4 weeks. Alternatively, strains may be stored at -80 indefinitely. This is preferable in that it reduces the likelihood of accumulating spontaneous mutations.

To make frozen stocks of yeast strains: -Use sterile technique and sterile solutions throughout this method.-

1. Grow a starter culture at 30 with shaking (250 rpm) until it reaches saturation.

2. In a 1.8 ml cryotube, mix 0.5 ml of the saturated culture with 0.5 ml of YPD containing 20% glycerol.

3. Flash freeze the tube in liquid nitrogen and store at -80.

4. To use, chip out a few pieces of the frozen stock using a sterile pipette tip or sterile toothpick and streak onto a plate containing the appropriate solid media.

5. Allow the yeast to grow at 30 until colonies appear (2-6 days).

10X YNB+AS

17 g/L YNB and 50 g/L AS

Dissolve 17 g Difco Yeast Nitrogen Base w/o Amino Acids and w/o Ammonium Sulfate and 50 g Ammonium Sulfate in 1L distilled water.

Autoclave for 15 min.

Store at room temp.

Yeast extract-Peptone-Dextrose medium: