Dandekar & Chandler:Selective media: Difference between revisions
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===Selective Antibiotics=== | ===Selective Antibiotics=== | ||
*Trimethoprim (for pseudomonas selection) | *Trimethoprim (for pseudomonas selection) | ||
| Line 32: | Line 46: | ||
''Do not combine LB and milk solutions before autoclaving unless you enjoy cleaning up large messes'' | ''Do not combine LB and milk solutions before autoclaving unless you enjoy cleaning up large messes'' | ||
''Do not forget to put a stir bar in/leave the stir bar in the skim milk autoclave bottle'' | |||
1. Prepare the following solutions: | 1. Prepare the following solutions: | ||
| Line 41: | Line 57: | ||
:* 300 ml H2O | :* 300 ml H2O | ||
:* 24 g powdered milk (2%) | :* 24 g powdered milk (2%) | ||
:- Stir well, leave stir bar in flask and autoclave | :- Stir well, '''leave stir bar in flask''' and autoclave | ||
2. Autoclave solutions separately | 2. Autoclave solutions separately | ||
Revision as of 11:46, 31 March 2014
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
Do not forget to put a stir bar in/leave the stir bar in the skim milk autoclave bottle
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
