Media

Materials

DMEM (Red Media)

Calf Serum

Pen-Strep

Fungizone

Media Prep

1. Obtain a bottle of DMEM from cold room, place in warm water bath
2. Obtain Calf Serum, Pen-Step and Fungizone from freezer (4° C), place in warm water bath
3. Add 50mL calf serum, 5mL Pen-Strep and 1mL Fungizone to DMEM
4. Place media in refrigerator for storage.

Filter using a 0.2micrometers filter bottle (500mL) :

- 450 mL of Dulbecco’s Modified Eagle Medium

- 50mL of Fœtal Bovine Serum (FBS)

- 425 mL of Knockout-DMEM medium (Gibco 10829)

- 75 mL ES-FBS

- 5mL Non-essential Amino Acid Solution

- 2.5 mL of L-glutamine

- 1 mL 2-mercaptoethanol

- 50 μL LIF

- 5 mL penicillin-streptomycin

Filter using a 0.2 μm Millipore Stericup filter into a 500 mL bottle

Plate ES cells on 0.2% gelatin-coated dishes

EB Medium

Pour into a filter (500mL bollte with 0.2 micrometers filter unit):

- 400mL of DMEM knockout Medium

- 5mL of non essential Amino Acid Solution

- 1mL of 2-mercapto ethanol

- 2.5mL of L-glutamine

- 100 mL of Knockout (KO)

M199 (500ml)

10% FBS (55 ml)

Fungizone (1ml/555ml media)

225mL DMEM

225mL Ham’s F12 medium

50mL FBS

5mL Pen-Strep