Dandekar & Chandler:Selective media

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(New page: Back Home: Dandekar & Chandler:Protocols ==Selective Media== ===Selective Antibiotics=== *Trimethoprim (for pseudomonas selection) ::Working concentration [100 ug/ml] from DMSO stock [...)
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===Protease Production===
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===Selection for Protease Production===
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Useful for selection of LasR mutants from WT Pseudomonas aeruginosa
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- Useful for selection of LasR mutants from WT Pseudomonas aeruginosa
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After competition against another species, first spread dilute co-culture onto antibiotic plate, then patch isolated pseudomonas onto milk plates.
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- After competition against another species, first spread dilute co-culture onto antibiotic plate and grow overnight, then patch isolated pseudomonas onto milk plates
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WT colonies will create clearings, allowing determination from LasR mutants.
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- WT colonies will create clearings, allowing determination from LasR mutants
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'''Skim Milk Plates'''
'''Skim Milk Plates'''
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''Do not combine LB and milk solutions before autoclaving -- they react and cause a giant mess!''
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*25% LB
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25% LB
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*4% milk
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4% milk
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*1.5% agar
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1.5% agar
 
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Directions:
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Directions
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''Do not combine LB and milk solutions before autoclaving unless you enjoy cleaning up large messes''
1. Prepare the following solutions:  
1. Prepare the following solutions:  
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:* 150 ml H2O
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:* 150 ml LB (can be previously autoclaved)
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:* 9 g bacto-agar
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:- Stir and autoclave; keep molten!
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* 150 ml H2O
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:* 300 ml H2O
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* 150 ml LB (can be previously autoclaved)
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:* 24 g powdered milk (2%)
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* 9g bacto-agar
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:- Stir well, leave stir bar in flask and autoclave
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Stir and autoclave; keep molten!
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-
 
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* 300 ml H2O
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* 24 g powdered milk (2%)
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Stir well, leave stir bar in flask and autoclave
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2.  Autoclave solutions separately
2.  Autoclave solutions separately

Revision as of 19:34, 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


Selection for 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 and grow overnight, then patch isolated pseudomonas onto milk plates

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


Skim Milk Plates

  • 25% LB
  • 4% milk
  • 1.5% agar


Directions:

Do not combine LB and milk solutions before autoclaving unless you enjoy cleaning up large messes

1. Prepare the following solutions:

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