Glycerol Stocks of E. Coli

Materials

 * LB plates (with appropriate selective drug for selection)
 * LB/SOC medium
 * Selective drug(s) for selection
 * Sterile Glycerol
 * 2 mL microcentrifuge tubes

Method

 * 1) Streak the original colony on a LB plate containing appropriate selective drug (e.g. 50 ug/mL ampicillin or 100 ug/mL kanamycin).
 * 2) Incubate the plate at 37 C overnight.
 * 3) Isolate a single colony and inoculate into 2-5 mL of LB containing appropriate selective drug.
 * 4) Grow the culture to mid-log phase by shaking at 225 rpm at 37 C. This usually takes 8-12 hours of growth (OD600 = 0.5-0.7).
 * 5) Pipette 0.85 mL of culture into microcentrifuge tubes. Add 0.15 mL of sterile glycerol. Use a large orifice pipette and draw and dispense glycerol slowly.
 * 6) Mix by inverting tubes 10-12X and store at -80 C.