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: xxul (xxul for double amount)
** 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 Cell Passaging E. coli Aliquots
|+ 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 || xxxxx || S-734 || Wild Type || All || None || Spring 2022
| Black || 1.25ul || S-734 || Wild Type || All || None || Spring 2022
|-
|-
| Green || 18.25ul || S-735 || Δppc || LB; glycerol; succinate; a-keto || sfGFP || Spring 2022
| Green || 1.83ul || 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
| Red || 1.42ul || S-736 || Δppc || LB; glycerol; succinate; a-keto || DSRed-Express2 || Spring 2022
|-
|-
| Blue || 51.28ul || S-737 || Δfbp || LB; glucose; galactose || sfGFP || Spring 2022
| Blue || 5.13ul || S-737 || Δfbp || LB; glucose; galactose || sfGFP || Spring 2022
|-
|-
| Orange || 11.55ul || S-738 || Δfbp || LB; glucose; galactose  || DSRed-Express2 || Spring 2023
| 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 xxx PFU of phage
# 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 xxul chloroform into 3 labeled microcentrifuge tubes. Label 3 additional tubes and set aside.
# 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, 3 minutes at max speed.
# 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

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% 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

Using E. coli Aliquots -- 10^7 total cells
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

  1. 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.
  2. Add cells
    • For WT passaging, this is 1.25ul of frozen, black, WT cell aliquots
  3. Incubate in shaking incubator for 1 hour
  4. Add 10^4 PFU of phage
  5. Incubate in shaking incubator for 23 hours
  6. Add additional sugars, if applicable
    • For WT passaging, add 389ul of 4% glucose
  7. Note whether flasks are clear or cloudy; add this results to document on Notability on lab iPad.
  8. Incubate in shaking incubator for 23-24 hours
  9. Label 6 microcentrifuge tubes.
  10. 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.
  11. Add 1ml of incubated cells + phage into each tube.
  12. Vortex for 15 seconds each.
  13. 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.
  14. 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.
  15. If the passage is dividable by 5, do a plaque assay to correct the number of added PFU for the next passage.
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