Media and Passaging
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Minimal Media Recipe
Final Volume | Water | 4x Minimal Salts | 2M MgSO4 | Trace Minerals (4000x) | Glucose (if needed) | |
---|---|---|---|---|---|---|
10ml | 7.5ml | 2.5ml | 10ul | 2.5ul | xxul | |
100ml | 75ml | 25ml | 100ul | 25ul | xxul | |
250ml | 180ml | 62.5ml | 250ul | 62.5ul | xxul |
- Glycerol should be added to a final concentration of xx% (or xx% if using a double amount).
If using:
- Black cells in 10ml: xxul (xxul for double amount)
- Green cells in 10ml: xxul (xxul for double amount)
- Red cells in 10ml: xxul (xxul for double amount)
- Green and Orange cells in 10ml: xxul (xxul for double amount)
- Red and Blue cells in 10ml: xxul (xxul for double amount)
Cell Aliquots
Aliquot Color | For 10^7 cells, use: | Strain Number | Genotype | Grows in: | Fluorescence | Frozen Date | |
---|---|---|---|---|---|---|---|
Black | xxxxx | S-734 | Wild Type | All | None | xxxx | |
Green | 18.25ul | S-735 | Δppc | LB; glycerol; succinate; a-keto | sfGFP | xxxx | |
Red | Example | S-736 | Δppc | LB; glycerol; succinate; a-keto | DSRed-Express2 | xxxx | |
Blue | Example | S-737 | Δfbp | LB; glucose; galactose | sfGFP | xxxx | |
Orange | 11.55ul | S-738 | Δfbp | LB; glucose; galactose | DSRed-Express2 | xxxx |
Phage Passaging Protocol
- Prepare 10ml of appropriate media
- Add cells
- Incubate in shaking incubator for 1 hour
- Add xxx PFU of phage
- Incubate in shaking incubator for 23 hours
- Add additional sugars, if applicable
- Incubate in shaking incubator for 23-24 hours
- Add xxul chloroform into 3 labeled microcentrifuge tubes. Label 3 additional tubes and set aside.
- Add 1ml of incubated cells + phage into each tube.
- Vortex for 15 seconds each.
- Put in microcentrifuge and spin down, 3 minutes at max speed.
- Pipet out 800ul into the previously labeled, empty tube. Put into phage box in the refrigerator.
- If the passage is dividable by 5, do a plaque assay to correct the number of added PFU for the next passage.