Preparation and using glycerol stocks of bacteria

Overview

This protocol describes how to prepare a glycerol stock of DH5a E. Coli and how to recover from the stock

Protocol

Preparing a glycerol stock

• Pick a colony and incubate in 3 ml LB broth overnight at 37ºC
• Place 920 µl of the 3 ml in a cryovial
• Add 80 µl sterile glycerol and vortex to mix
• Store at -80ºC indefinitely

Recovering from a glycerol stock

• Method 1: Thaw to room temp, dab with a sterile inoculating loop. Streak on agar plate with appropriate antibiotic. To ensure appropriate density, streak densely on one-third of plate, less dense on one-third, and sparsely on remaining one-third of plate. Incubate plates overnight at 37ºC and pick colonies the next day.
• Method 2: Scrape frozen surface of culture with sterile inoculating loop and streak as above.
• Method 3: Scrape frozen surface of culture with sterile pipette tip. Place pipette tip in 2 ml growth media and incubate overnight at 37ºC in a shaker.