Media and Passaging: Difference between revisions
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*Glycerol should be added to a final concentration of xx% (or xx% if using a double amount). | *Glycerol should be added to a final concentration of xx% (or xx% if using a double amount). | ||
If using: | If using: | ||
** Black cells in 10ml: | ** Black cells in 10ml: 1.25ul (for 10^7 cells total) | ||
** Green 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) | ** Red cells in 10ml: xxul (xxul for double amount) | ||
** Green and Orange 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) | ** Red and Blue cells in 10ml: xxul (xxul for double amount) | ||
==Cell Aliquots== | ==Cell Aliquots== | ||
{| class="wikitable" style="margin:auto" | {| class="wikitable" style="margin:auto" | ||
|+ Using | |+ Using E. coli Aliquots -- 10^7 total cells | ||
|- | |- | ||
! 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 || | | Black || 1.25ul || S-734 || Wild Type || All || None || Spring 2022 | ||
|- | |- | ||
| Green || | | Green || 1.83ul || S-735 || Δppc || LB; glycerol; succinate; a-keto || sfGFP || Spring 2022 | ||
|- | |- | ||
| Red || | | Red || 1.42ul || S-736 || Δppc || LB; glycerol; succinate; a-keto || DSRed-Express2 || Spring 2022 | ||
|- | |- | ||
| Blue || | | Blue || 5.13ul || S-737 || Δfbp || LB; glucose; galactose || sfGFP || Spring 2022 | ||
|- | |- | ||
| Orange || | | Orange || 1.16ul || S-738 || Δfbp || LB; glucose; galactose || DSRed-Express2 || Spring 2023 | ||
|} | |} | ||
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# Prepare 10ml of appropriate media | # Prepare 10ml of appropriate media | ||
#* For WT passaging, this is 10ml of MM + 0.3% glucose for '2x' lineages and 10ml of MM + 0.15% glucose for '1x' lineages. | |||
# Add cells | # Add cells | ||
#* For WT passaging, this is 1.25ul of frozen, black, WT cell aliquots | |||
# Incubate in shaking incubator for 1 hour | # Incubate in shaking incubator for 1 hour | ||
# Add | # Add 10^4 PFU of phage | ||
# Incubate in shaking incubator for 23 hours | # Incubate in shaking incubator for 23 hours | ||
# Add additional sugars, if applicable | # Add additional sugars, if applicable | ||
#* For WT passaging, add 389ul of 4% glucose | |||
# Note whether flasks are clear or cloudy; add this results to document on Notability on lab iPad. | |||
# Incubate in shaking incubator for 23-24 hours | # Incubate in shaking incubator for 23-24 hours | ||
# Add | # Label 6 microcentrifuge tubes. | ||
# Add 125ul chloroform into a labeled microcentrifuge tube. Ensure picking chloroform from the bottom of the tube, as there is a water layer on top. | |||
# Add 1ml of incubated cells + phage into each tube. | # Add 1ml of incubated cells + phage into each tube. | ||
# Vortex for 15 seconds each. | # Vortex for 15 seconds each. | ||
# Put in microcentrifuge and spin down, | # Put in microcentrifuge and spin down, 5 minutes at max speed. You should see 2-3 layers of liquid, with the middle layer (if present) being a thin line of dead cells. If you only see 1 layer, something is off -- probably the top layer of water was added from the chloroform. Please start over. | ||
# Pipet out 800ul into the previously labeled, empty tube. Put into phage box in the refrigerator. | # Pipet out 800ul into the previously labeled, empty tube. Put into phage box in the refrigerator. | ||
#* For WT passaging, create a 10^-2 dilution for the next phage passage by adding 990ul PBS to 10ul of collected, chloroform-purified phage. | |||
# If the passage is dividable by 5, do a plaque assay to correct the number of added PFU for the next passage. | # If the passage is dividable by 5, do a plaque assay to correct the number of added PFU for the next passage. |
Latest revision as of 12:23, 8 January 2024
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: 1.25ul (for 10^7 cells total)
- 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 | 1.25ul | S-734 | Wild Type | All | None | Spring 2022 | |
Green | 1.83ul | S-735 | Δppc | LB; glycerol; succinate; a-keto | sfGFP | Spring 2022 | |
Red | 1.42ul | S-736 | Δppc | LB; glycerol; succinate; a-keto | DSRed-Express2 | Spring 2022 | |
Blue | 5.13ul | S-737 | Δfbp | LB; glucose; galactose | sfGFP | Spring 2022 | |
Orange | 1.16ul | S-738 | Δfbp | LB; glucose; galactose | DSRed-Express2 | Spring 2023 |
Phage Passaging Protocol
- Prepare 10ml of appropriate media
- For WT passaging, this is 10ml of MM + 0.3% glucose for '2x' lineages and 10ml of MM + 0.15% glucose for '1x' lineages.
- Add cells
- For WT passaging, this is 1.25ul of frozen, black, WT cell aliquots
- Incubate in shaking incubator for 1 hour
- Add 10^4 PFU of phage
- Incubate in shaking incubator for 23 hours
- Add additional sugars, if applicable
- For WT passaging, add 389ul of 4% glucose
- Note whether flasks are clear or cloudy; add this results to document on Notability on lab iPad.
- Incubate in shaking incubator for 23-24 hours
- Label 6 microcentrifuge tubes.
- Add 125ul chloroform into a labeled microcentrifuge tube. Ensure picking chloroform from the bottom of the tube, as there is a water layer on top.
- Add 1ml of incubated cells + phage into each tube.
- Vortex for 15 seconds each.
- Put in microcentrifuge and spin down, 5 minutes at max speed. You should see 2-3 layers of liquid, with the middle layer (if present) being a thin line of dead cells. If you only see 1 layer, something is off -- probably the top layer of water was added from the chloroform. Please start over.
- Pipet out 800ul into the previously labeled, empty tube. Put into phage box in the refrigerator.
- For WT passaging, create a 10^-2 dilution for the next phage passage by adding 990ul PBS to 10ul of collected, chloroform-purified phage.
- If the passage is dividable by 5, do a plaque assay to correct the number of added PFU for the next passage.