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Protocol 2: 5 mL Overnight

What you will need:

  • 10 mL culture tube Use 16mm x 160mm or 16mm x 125 mm
  • 5 mL LB
  • concentrated antibiotics
  • Single colonies on a plate (Best not to start O/N from glycerol stocks)


  • Pipet 1.5-20uL of the appropriate antibiotic to the appropriate working concentration into culture tube.
  • Add 5 mL non-contaminated LB. Do this first, then add antibiotic
  • Select single colony using a sterile instrument that has been cooled and inoculate the liquid culture.
  • Place culture tube in incubator at 37 C overnight shaking vigorously (250 rpm).


For I0500, culture overnight; then add 1 mM ITPG in morning and continue culture for another 3 hrs. Then miniprep.