Dandekar & Chandler:Selective media

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Revision as of 19:08, 22 August 2013

Back Home: Dandekar & Chandler:Protocols

Selective Media

Selective Antibiotics

  • Trimethoprim (for pseudomonas selection)
Working concentration [100 ug/ml] from DMSO stock [100 mg/ml]
  • Triple antibiotic (for burkholderia selection from co-culture)
150,000 IUs/L Polymixin B (=17.3 mg/L)
5 mg/L gentamicin
100 mg/L ticarcillin


Protease Production

Useful for selection of LasR mutants from WT Pseudomonas aeruginosa

After competition against another species, first spread dilute co-culture onto antibiotic plate, then patch isolated pseudomonas onto milk plates.

WT colonies will create clearings, allowing determination from LasR mutants.


Skim Milk Plates

Do not combine LB and milk solutions before autoclaving -- they react and cause a giant mess!

25% LB

4% milk

1.5% agar


Directions

1. Prepare the following solutions:

  • 150 ml H2O
  • 150 ml LB (can be previously autoclaved)
  • 9g bacto-agar

Stir and autoclave; keep molten!

  • 300 ml H2O
  • 24 g powdered milk (2%)

Stir well, leave stir bar in flask and autoclave

2. Autoclave solutions separately

3. Start milk solution stirring on stir plate

4. Slowly pour molten LB agar into milk solution

Recipe above pours one sleeve of plates

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