IGEM:Virginia 2012/Protocols/SSM Media Preparation: Difference between revisions

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Line 18: Line 18:
| CaCl<sub>2</sub>  2H<sub>2</sub>O  ''*20 mg/mL'' || 20mg || 40mg || 80mg || 100mg
| CaCl<sub>2</sub>  2H<sub>2</sub>O  ''*20 mg/mL'' || 20mg || 40mg || 80mg || 100mg
|-
|-
| Q.S. with deionized water to** || 980mL || 1960mL || 3920mL || 4900mL
| Bring to Volume with deionized water to** || 980mL || 1960mL || 3920mL || 4900mL
|}
|}


Line 26: Line 26:
  *Note: if pouring blood plates, bring volume up to 100 mL/L '''LESS''' than above.
  *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.
  *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 H<sub>2</sub>O
*0.80g L-cystine
*A Vortex
*200 mL deionized H<sub>2</sub>O
{| border="0" cellpadding="0"
|-
| width="50 pt" | FeSO<sub>4</sub> || width="50 pt" | 0.2g
|-
| ascorbic acid || 0.4g
|-
| nicotinic acid (niacin) || 80 mg
|-
| glutathione; reduced form || 2.0g
|}


==Procedure==
==Procedure==

Revision as of 11:47, 31 May 2012

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
FeSO4 0.2g
ascorbic acid 0.4g
nicotinic acid (niacin) 80 mg
glutathione; reduced form 2.0g

Procedure

  • 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.

Notes

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


References

Contact

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