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From 2013.igem.org

• Add (35 g/L of LB Agar) or (25 g/L LB broth and 15 g/L Agar) to a flask with distilled water

• Dissolve and autoclave for 30 mins (Basement lab autoclave machine) or 1 hour ( Barker lab autoclave machine)

• Antibiotics should be added at the correct concentration and volume when the agar has cooled to around 50°C or cool enough for hands to be touching the exterior of the flask.

• Concentrations of antibiotics:
  • Chloramphenicol 34 µg/µL - Blue
  • Kanamycin 50 µg/µL - Green
  • Ampicillin 100 µg/µL - Red

• For every mL of LB agar solution, add 1 µL of antibiotics.
  • Since I was using 300 mL of water, I added .3 mL of kanamycin

• The plates are poured and cooled, then marked with a marker to signify the antibiotic.

• Agar stabs are from the same protocol, just poured into tubes and capped.

• Media solution is made from LB broth (25 g/L), autoclaved, and antibiotics added to it.

• From the toothpick used to create an agar stab, the toothpick is placed into the media solution to start the culture.