IGEM:Virginia 2012/Protocols/SSM Media Preparation: Difference between revisions
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*Note: *Make stock solutions and add this amount. | *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: **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: 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: 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. | ||
==Procedure== | ==Procedure== | ||
Revision as of 11:35, 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 |
| Q.S. 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.
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
- Who has experience with this protocol?
or instead, discuss this protocol.
