Help:How to make agar
- Prepare LB agar according to manufacturer's instructions (e.g. 35g of agar in 1L diH2O for Difco LB Lennox Agar). Adjust pH to 7.5 if necessary with 1M NaOH.
- Autoclave media (with caps loosened!) for 20 minutes at 121 degrees
- Cool down to 55 degrees. Since autoclaved agar cools down slowly, a good way to do this is prepare and autoclave it in the morning then leave it in a 55 degree water bath. Don't try to cool it down quickly because the glass may shatter.
- Once it's cool enough (50-60 degrees) - add antibiotics: 1mL per litre for Ampicillin, Chloramphenicol, Kanamycin and 3mL per litre for Tetracycline. (concentrations of liquid stocks: Amp- 100mg/ml, Cm- 35mg/ml, Kan- 50mg/ml, Tet- 5mg/ml.)
- Swirl to mix; try not to make many bubbles.
- Fill petri dishes or screw-cap tube half-full with agar and cap. Once set, store in refrigerator for future use.
- Don't forget to label the plates/tubes with the appropriate antibiotic!
- We have a standard color key for our antibiotics:
- Orange denotes ampicillin,
- Red denotes kanamycin,
- Green denotes chloramphenicol,
- Yellow denotes tetracycline.