TABLE 1.
Step | Ingredient or procedure | Amt |
---|---|---|
Base solution | KH2PO4 | 0.5 g |
Sodium glutamate | 10.72 g | |
NaCl | 2.5 g | |
KCl | 0.2 g | |
MgCl2 · 6H2O | 0.1 g | |
Tris | 1.525 g | |
α-Ketoglutamic acid | 2.0 g | |
Sodium pyruvate | 2.0 g | |
CaCl2 | 20.0 mg | |
Deionized H2O | To 909.0 ml | |
Adjust to pH 7.8 with NaOH, autoclave, and store at 4°C | ||
CDM supplement | Ascorbic acid | 0.2 g |
Glutathione, reduced | 1.0 g | |
Deionized H2O | To 100.0 ml | |
Store at −20°C | ||
l-Cystine supplement | Deionized H2O | 3.0 ml |
HCl (11.6 M) | 1.0 ml | |
l-Cystine | 0.4 g | |
Deionized H2O | To 100.0 ml | |
Store at −20°C | ||
l-Proline supplement | l-Proline | 3.0 g |
Deionized H2O | To 200.0 ml | |
Autoclave and store at −20°C | ||
l-Phenylalanine supplement | l-Phenylalanine | 4.0 g |
Deionized H2O | To 400.0 ml | |
Autoclave and store at 4°C | ||
Vitamins | p-Aminobenzoic acid | 10 mg |
Biotin | 1 mg | |
Folic acid | 10 mg | |
Pyridoxyl HCl | 80 mg | |
Riboflavin | 40 mg | |
Thiamine HCl | 40 mg | |
Calcium pantothenate | 80 mg | |
Nicotinamide | 200 mg | |
Deionized H2O | 800 ml | |
Add 2.5 N NaOH until solution clears | ||
Add deionized H2O to a final volume of 1,000 ml | ||
Store at −20°C | ||
Prepare final medium | Base medium | 909 ml |
l-Cystine stock | 10 ml | |
l-Proline stock | 16 ml | |
CDM supplement | 10 ml | |
l-Phenylalanine stock | 40 ml | |
Vitamins | 10 ml | |
Sterilize by ultrafiltration (0.2-μm pore size) |
CDM may be stored for up to 2 weeks at 4°C. Stock solutions may be stored for up to 3 months.