IGEM:Virginia 2012/Protocols/SSM Media Preparation
< IGEM:Virginia 2012 | Protocols
|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|
|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.
- 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
- 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.
- 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:
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