Pelling:Protocols/Agar Plates

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Agar Bacteriological Plates

  1. Need a ratio of 25g LB Broth : 15g Agar : 1L dH2O (For 400mL of dH2O, you need 10g LB, 6g Agar)
  2. Combine in a 500mL glass Duran bottle with a stir bar
  3. Autoclave in wet cycle for 20 minutes
  4. Retrieve immediately!
  5. Stir at 200rpm for 15 minutes without any heating. You want the solution to cool to about 50°C.
  6. Once cooled enough to keep your bare palm on the bottle without pain, add antibiotic to a final concentration of:
    • Kanamycin at 50μg/mL
    • Ampicillin 100μg/mL
    • Note: Use or make a 1000X stock solution in dH2O and store in the fridge.
    • IMPORTANT we use 1000X stock solutions. Therefore in a 400mL bottle of LB:Agar you put in 400μL of antibiotics!!!!
  7. Pour liquid into sterile 100mm bacterial culture plates (NOT the 100 mm mammalian cell culture plates) to a thickness of about 0.5-1.0 cm, ensuring absolutely NO bubbles remain
  8. Mark the bottom of the plate with K or A for antibiotic
  9. Let plates set for a few hours or overnight on bench top (fume hood will dry them out).
  10. Place them in a bag and store in fridge up to about 3 months. Try to store upside down (bottom facing up) to prevent dehydration.