Proulx:Media Prep

Overview

A collection of fundamental protocols for media preparation for the Proulx lab at UCSB.

Plate Pouring Protocol

Materials

• Premix Media Powder
• Difco agar
• Distilled water
• 2 L flask
• Foil
• Autoclave tape
• Plastic tray
• Petri dishes

Procedure

1. Measure 1 L distilled water
2. Add half the water and a stirring magnet to a 2 L bottle or flask
3. Place bottle on stirrer
4. Add powder mix from recipe for the media you are preparing. Use a funnel to pour in the powder and ensure that the powder does not stick to the flask above the water line.
5. Let stir in for a minute
6. Add the remaining water
7. Make a foil cover for the flask and fit it to the flask. This will be autoclaved off the mouth of the flask but taped to it so that it can be covered when removed from the autoclave. Use autoclave tape.
8. Place flasks in a plastic autoclave tray; fill tray with about 1 inch of water
9. Use the cart to transport to the 4th floor autoclave and bring the orange autoclave mittens
10. Place tray in the autoclave; record date and start time on the sign-in sheet; Use the liquid cycle for 35 minutes
11. Remove tray from autoclave, fit the foil over the flask mouth.
12. Let agar cool to ~55°C (you should be able to touch the jar for at least five seconds)
13. Remove sterile Petri dishes from plastic bag (save the bag for storage).
14. Label plates with the media type and date.
15. Pour a thin layer of mixture (~20mL) into each plate, being careful to not lift the cover off excessively (you should be able to just open up enough to pour. If necessary, swirl plate to distribute agar on bottom completely.
16. Let each plate cool until it is solid (~20 minutes), then flip so as to avoid condensation on the agar. Let plates dry overnight (or longer) on bench.
17. Store plates in plastic bags in plate fridge by fume hood.

Recipes

The premix-powders are very fine and lightweight. It is important to minimize the spread of dust from this powder as it is corrosive to electronic balances and generally makes a sticky mess. Put plastic wrap over the scale before zeroing and weighing and gently scoop the powder and place on the weigh boats.

YPD

Recipe for 1 L of media:

1. 50 g Difco YPB broth powder
2. 15 g Difco agar

SD

Synthetic Defined Media, dropout version. This uses an additive to include most, but not all, AAs.

Recipe for 1 L of media:

1. 20 g Dextrose
2. 6.8 g Yeast Nitrogen Base w/out amino acids
3. 0.55 g Sunrise CSM-His-Leu-Lys-Trp-Ura
4. 15 g Difco agar

Sigma Y2001 Synthetic Media (missing His, Leu, Tryp, Ura)

Recipe for 1 L of media:

1. 20 g Dextrose
2. 6.8 g Yeast Nitrogen Base w/out amino acids (Sigma Y0626)
3. 1.46 g Yeast Synthetic Drop-out Medium Supplements (Sigma Y2001)
4. 15 g Difco agar

USBiological Drop-out mix (missing His, Leu, Lysine, Cysteine, Ura)

Recipe for 1 L of media:

1. 20 g Dextrose
2. 6.8 g Yeast Nitrogen Base w/out amino acids (Sigma Y0626)
3. 1.3 g Drop-out mix (USBio D9517-06A)
4. 15 g Difco agar

LB

Recipe for 1 L of media:

1. 20 g LB premix powder (Sigma)
2. 15 g Difco agar

Single Amino Acid Stock Solutions

To make single amino acid stock solutions use the following table for how much to add to 100 ml of H20

Amino Acid Table

Amino Acid	per 100 ml	required final concentration
Leucine	720 mg	380 mg/L
Lysine	360 mg	76 mg/L
Uracil	240 mg	76 mg/L
Methionine	240 mg	76 mg/L