Dandekar & Chandler:Pseudomonas Mating: Difference between revisions
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== Pseudomonas Mating Protocol == | |||
''Master compilation of protocols from Sudha, Thao and Brook - Assembled by Moe T.'' | |||
Before starting, make sure you have all the appropriate plates prepared: | |||
::LB | |||
::LB + antibiotic required for deletion construct (needed for multiple days) | |||
::Pseudomonas isolation agar (PIA) + antibiotic (needed for multiple days) | |||
::No Salt LB + 10% sucrose (needed for multiple days) | |||
Pseudomonas requires higher concentrations of antibiotic than what E. coli needs to maintain a plasmid; for gentamicin | |||
===Day 1=== | |||
Transform E. coli S17-1 with deletion construct(s) needed to make the knockout. **S17-1 has ''tra'' genes required for conjugation integrated on the chromosome** | |||
===Day 2=== | |||
Streak S17-1 with construct to LB+ab for singles and leave at 37°C O/N | |||
===Day 3=== | |||
Grow PAO1 in LB and S17-1 with construct in '''LB+ab''' tubes overnight at 37°C | |||
===Day 4=== | |||
Dilute the S17-1 culture 1:100 in a tube with 5mL '''LB+ab''' and grow at 37°C to mid-log phase (OD600 ~0.3-0.6) with shaking | |||
Dilute PAO1 into 50 mL flask 1:10 with 10mL LB and place at 42°C* with '''NO shaking''' to increase the frequency of recombination of foreign DNA *''This is the small incubator by heat blocks in EPG lab'' | |||
When S17-1 reaches OD ~0.3-0.6, gently mix cultures at 1:1 and 3:1 (donor: recipient) in 15mL conical tubes; spin at 2000 rpm at RT for 10 minutes | |||
Remove supernatant and resuspend each pellet in 40 μL LB | |||
Spot each culture mix onto LB agar plates with no antibiotic; incubate the plate O/N at 30°C* *''This is the bottom incubator in EPG lab'' | |||
===Day 5=== | |||
Scrape the cells from the spot and resuspend in 1 mL 1x PBS | |||
Plate onto '''PIA+ab''': | |||
::100 μL of suspension directly | |||
::100 μL of 1:10 dilution | |||
::100 μL of 1:100 dilution | |||
::The remaining pellet of cells ''(This always makes a lawn for me, so I omit – Nicole)'' | |||
Incubate O/N at 30°C | |||
===Day 6/7=== | |||
''Colonies may be small – either go another 24 hrs at 30°C or transfer to 37°C in the morning and colonies should be large enough by end of day'' | |||
Patch colonies that appear on the PIA+ab plates again to '''PIA+ab''' to confirm single cross integration of plasmid onto PAO1 chromosome | |||
Incubate O/N at 30°C | |||
===Day 8=== | |||
Patch colonies again to '''LB+ab''' to confirm single cross integration of plasmid backbone onto the chromosome |
Revision as of 10:34, 20 February 2014
Back to Protocols
Pseudomonas Mating Protocol
Master compilation of protocols from Sudha, Thao and Brook - Assembled by Moe T. Before starting, make sure you have all the appropriate plates prepared:
- LB
- LB + antibiotic required for deletion construct (needed for multiple days)
- Pseudomonas isolation agar (PIA) + antibiotic (needed for multiple days)
- No Salt LB + 10% sucrose (needed for multiple days)
Pseudomonas requires higher concentrations of antibiotic than what E. coli needs to maintain a plasmid; for gentamicin
Day 1
Transform E. coli S17-1 with deletion construct(s) needed to make the knockout. **S17-1 has tra genes required for conjugation integrated on the chromosome**
Day 2
Streak S17-1 with construct to LB+ab for singles and leave at 37°C O/N
Day 3
Grow PAO1 in LB and S17-1 with construct in LB+ab tubes overnight at 37°C
Day 4
Dilute the S17-1 culture 1:100 in a tube with 5mL LB+ab and grow at 37°C to mid-log phase (OD600 ~0.3-0.6) with shaking
Dilute PAO1 into 50 mL flask 1:10 with 10mL LB and place at 42°C* with NO shaking to increase the frequency of recombination of foreign DNA *This is the small incubator by heat blocks in EPG lab
When S17-1 reaches OD ~0.3-0.6, gently mix cultures at 1:1 and 3:1 (donor: recipient) in 15mL conical tubes; spin at 2000 rpm at RT for 10 minutes
Remove supernatant and resuspend each pellet in 40 μL LB
Spot each culture mix onto LB agar plates with no antibiotic; incubate the plate O/N at 30°C* *This is the bottom incubator in EPG lab
Day 5
Scrape the cells from the spot and resuspend in 1 mL 1x PBS Plate onto PIA+ab:
- 100 μL of suspension directly
- 100 μL of 1:10 dilution
- 100 μL of 1:100 dilution
- The remaining pellet of cells (This always makes a lawn for me, so I omit – Nicole)
Incubate O/N at 30°C
Day 6/7
Colonies may be small – either go another 24 hrs at 30°C or transfer to 37°C in the morning and colonies should be large enough by end of day Patch colonies that appear on the PIA+ab plates again to PIA+ab to confirm single cross integration of plasmid onto PAO1 chromosome Incubate O/N at 30°C
Day 8
Patch colonies again to LB+ab to confirm single cross integration of plasmid backbone onto the chromosome