IGEM:Virginia 2012/Protocols/SSM Media Preparation

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Contents

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:

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!


References

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