Dandekar & Chandler:Pa/Bc competition setup

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(New page: Back to Dandekar & Chandler:Protocols ==Coculture competition protocol==)
(Coculture competition protocol)
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Back to [[Dandekar & Chandler:Protocols]]
Back to [[Dandekar & Chandler:Protocols]]
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==Coculture competition protocol==
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==Pseudomonas:Burkholderia co-culture competition protocol==
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'''Two days before:'''
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Streak strains of interest from frozen stock
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::- One parent Pa, one QS mutant Pa, one competitive Bc
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::- Streak onto DRY plates to ensure single colonies for inoculation
 +
 
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'''Morning before:'''
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::- Using single colonies from overnight plates, inoculate 3 ml LB + 50mM MOPS in 16mm tubes
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::- Grow ~6-8hrs
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'''Evening before:'''
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::- From morning culture, make serial dilutions to ensure there is < 0.5 OD tube for the next morning
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:::For consistency between experiments, it’s important to make sure that your starting culture hasn’t turned on QS; Once cells have entered stationary phase, and become QS-on, they will continue to be QS-on even with back diluting
 +
:::Using 3 ml LB, set up 7 tubes per culture; make 1st tube a 10X dilution, and continue 10x dilutions through the series
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::- If dilutions are made at 5pm, 9 am is 16 hours of growth; check tubes immediately upon arriving to lab
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'''Morning of:'''
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::- Check dilution tubes 5-7; hopefully one will be ≤ OD 0.5
 +
::- Cells above OD 0.6 may already be QS-on; discard those tubes
 +
::- If cells are in the 0.1-0.2 OD range, let them continue to grow while setting up 96-well plate and antibiotic agar plates are set up (check on them ~30 min later)

Revision as of 17:31, 20 August 2013

Back to Dandekar & Chandler:Protocols

Pseudomonas:Burkholderia co-culture competition protocol

Two days before:

Streak strains of interest from frozen stock

- One parent Pa, one QS mutant Pa, one competitive Bc
- Streak onto DRY plates to ensure single colonies for inoculation

Morning before:

- Using single colonies from overnight plates, inoculate 3 ml LB + 50mM MOPS in 16mm tubes
- Grow ~6-8hrs

Evening before:

- From morning culture, make serial dilutions to ensure there is < 0.5 OD tube for the next morning
For consistency between experiments, it’s important to make sure that your starting culture hasn’t turned on QS; Once cells have entered stationary phase, and become QS-on, they will continue to be QS-on even with back diluting
Using 3 ml LB, set up 7 tubes per culture; make 1st tube a 10X dilution, and continue 10x dilutions through the series
- If dilutions are made at 5pm, 9 am is 16 hours of growth; check tubes immediately upon arriving to lab

Morning of:

- Check dilution tubes 5-7; hopefully one will be ≤ OD 0.5
- Cells above OD 0.6 may already be QS-on; discard those tubes
- If cells are in the 0.1-0.2 OD range, let them continue to grow while setting up 96-well plate and antibiotic agar plates are set up (check on them ~30 min later)
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