IGEM:Virginia 2012/Protocols/SSM Media Preparation

Materials

BASAL MEDIUM to make	1 Liter	2 Liters	4 Liters	5 Liters
Glutamic acid, monosodium salt, 1H2O	10.72g	21.44g	42.88g	53.60g
Trisma Base	1.525g	3.05g	6.10g	7.63g
NaCl	2.50g	5.00g	10.00g	12.50g
KH2PO4	0.50g	1.00g	2.00g	2.50g
KCl *200 mg/mL	0.20g	0.40g	0.80g	1.00g
MgCl2 6H2O *100mg/mL (0.5 mM)	100mg	200mg	400mg	500mg
CaCl2 2H2O *20 mg/mL	20mg	40mg	80mg	100mg
Bring to Volume with deionized water to**	980mL	1960mL	3920mL	4900mL

• Note: *Make stock solutions and add this amount.
• Note: **These Volumes are to allow for 20 mL/L worth of supplements to be added later.

*Note: For SSM-S, add 2.0 g/L of 2-ketoglutarate and pyruvate before pH'ing.
*Note: if pouring blood plates, bring volume up to 100 mL/L LESS than above.
*Note: it is convenient to make up the last three salts in a concentrated stock solution, such that you can use 1mL/liter of SSM.

SSM Supplement

• 2 mL conc. HCL
• 6 mL distilled H₂O
• 0.80g L-cystine
• A Vortex
• 200 mL deionized H₂O
• 0.2g FeSO₄
• 0.4g of ascorbic acid
• 80mg of nicotinic acid (niacin)
• 2.0g of glutathione; reduced form

Procedure

Basal Medium

• Adjust to pH 7.60 with concentrated HCl (about 0.5-0.7 mL/L).
• Autoclave for 20 min. (May store at this stage at 4°C as "SSM Incomplete.")'
• After cooling and before use, add 10 mL/L of both proline and SSM supplements (see below).

*Note: for SSM-S, also add 1 mL/L SSM-S supplement. May store basal medium at 4°C for short periods of time and at -20°C for somewhat longer.

SSM Supplement

• Mix 2 mL conc. HCl and 6 mL distilled H₂O.
• Add 0.80g L-cystine.
• Vortex to dissolve.
• Bring to volume 200 mL with deionized H₂O. May need to add a little more HCl at this point if cystine comes out of solution.
• Then add the following:
  • 0.2g FeSO₄
  • 0.4g of ascorbic acid
  • 80mg of nicotinic acid (niacin)
  • 2.0g of glutathione; reduced form
• Filter sterilize, and make 10 mL aliquots. Store at -20°C.

Notes

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