Creating Bacterial Glycerol Stocks

Collect all required materials including a 2 mL screw cap cryovial, 50% glycerol solution, overnight bacterial culture, pipette with tips, 70% isopropanol, sterile loop or toothpicks, dry ice, and an LB agar plate.

Dilute 100% glycerol in dH₂O to create a 50% glycerol stock solution for use in the glycerol stocks.

Use a marker to write labels directly onto the 2 mL screw cap tubes before freezing, as frozen tubes are difficult to label and labels unstick at low temperatures.

Add 500 μL of 50% glycerol solution and 500 μL of overnight bacterial culture to a labeled 2 mL screw top cryovial, achieving a final glycerol concentration of 25%. Shake the tube 5-6 times until completely mixed and uniform.

Place the mixed glycerol stock tube in a -80°C freezer where it will remain stable for years. Avoid multiple freeze-thaw cycles to preserve long-term shelf life.

Remove the glycerol stock from the freezer and use a sterile loop, toothpick, or pipette tip to scrape frozen bacteria from the top without thawing. Keep the tube on dry ice while streaking to preserve shelf life.

Streak the recovered bacteria onto an LB agar plate and grow overnight at the appropriate temperature specified on the plasmid's information page.

The next day, use the grown bacterial colonies to start an overnight culture for plasmid DNA preparation.