Dandekar & Chandler:Selective media

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Back Home: [[Dandekar & Chandler:Protocols]]
 
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==Selective Media==
 
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===Selective Antibiotics===
 
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*Trimethoprim (for pseudomonas selection)
 
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::Working concentration [100 ug/ml] from DMSO stock [100 mg/ml]
 
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*Triple antibiotic (for burkholderia selection from co-culture)
 
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::150,000 IUs/L Polymixin B (=17.3 mg/L)
 
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::5 mg/L gentamicin
 
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::100 mg/L ticarcillin
 
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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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- 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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'''Skim Milk Plates'''
 
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*25% LB
 
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*4% milk
 
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*1.5% agar
 
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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''
 
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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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:* 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
 
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3.  Start milk solution stirring on stir plate
 
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4. Slowly pour molten LB agar into milk solution
 
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''Recipe above pours one sleeve of plates''
 

Revision as of 16:49, 11 October 2013

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