Overnight Cultures

Reagents and Materials (per culture prepared):

  • 10mL culture tube. Use 16mm x 160mm or 16mm x 125mm
  • 5mL LB
  • 5μL 1000X antibiotics
  • Single colonies on a plate (best not to start an overnight from a glycerol stock)


  1. Add 3mL sterile/autoclaved LB in a 15mL Falcon tube
  2. Pipet 3μL of 1000X antibiotic into the LB
  3. Select a single colony using a sterile toothpick or pipette tip
  4. Place toothpick or pipette tip in the culture tube and stir
  5. Remove toothpick, or in the case of a pipette tip, leave in the tube
  6. Place culture tube in incubator at 37 degrees Celsius overnight shaking vigorously (250 RPM)