Dandekar & Chandler:Pseudomonas Mating

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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 13:34, 20 February 2014

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Contents

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

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