Media and Passaging: Difference between revisions
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EricMiller (talk | contribs) (Created page with "==Minimal Media Recipe== {| class="wikitable" style="margin:auto" |+ LeMaster-Richards Minimal Media (LeMaster and Richards, 1982) |- ! 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%...") |
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! Aliquot Color !! For 10^7 cells, use: !! Strain Number !! Genotype !! Grows in: !! Fluorescence !! Frozen Date !! | ! Aliquot Color !! For 10^7 cells, use: !! Strain Number !! Genotype !! Grows in: !! Fluorescence !! Frozen Date !! | ||
|- | |- | ||
| Black || xxxxx || S-734 || Wild Type || All || None || | | Black || xxxxx || S-734 || Wild Type || All || None || Spring 2022 | ||
|- | |- | ||
| Green || 18.25ul || S-735 || Δppc || LB; glycerol; succinate; a-keto || sfGFP || | | Green || 18.25ul || S-735 || Δppc || LB; glycerol; succinate; a-keto || sfGFP || Spring 2022 | ||
|- | |- | ||
| Red || | | Red || 14.16ul || S-736 || Δppc || LB; glycerol; succinate; a-keto || DSRed-Express2 || Spring 2022 | ||
|- | |- | ||
| Blue || | | Blue || 51.28ul || S-737 || Δfbp || LB; glucose; galactose || sfGFP || Spring 2022 | ||
|- | |- | ||
| Orange || 11.55ul || S-738 || Δfbp || LB; glucose; galactose || DSRed-Express2 || | | Orange || 11.55ul || S-738 || Δfbp || LB; glucose; galactose || DSRed-Express2 || Spring 2023 | ||
|} | |} | ||
==Phage Passaging Protocol== | ==Phage Passaging Protocol== |
Revision as of 12:45, 17 September 2023
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 | Spring 2022 | |
Green | 18.25ul | S-735 | Δppc | LB; glycerol; succinate; a-keto | sfGFP | Spring 2022 | |
Red | 14.16ul | S-736 | Δppc | LB; glycerol; succinate; a-keto | DSRed-Express2 | Spring 2022 | |
Blue | 51.28ul | S-737 | Δfbp | LB; glucose; galactose | sfGFP | Spring 2022 | |
Orange | 11.55ul | S-738 | Δfbp | LB; glucose; galactose | DSRed-Express2 | Spring 2023 |
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.