Preparing Cryo stocks

Preparation of glycerol stocks

E. coli strains can be stored for many years at –70°C in medium containing 15% glycerol. Prepare glycerol stocks of bacteria as follows:

1. Add 0.15 ml glycerol (100%) to a 2 ml screw-cap vial and sterilize by autoclaving. Vials of sterilized glycerol can be prepared in batches and stored at room temperature until required.

2. Add 0.85 ml of a logarithmic-phase E. coli culture (see “Growth of E. coli cultures”, page 7) to the vial of pre-sterilized glycerol.

3. Vortex the vial vigorously to ensure even mixing of the bacterial culture and the glycerol.

4. Freeze in an dry ice–ethanol bath or liquid nitrogen and store at –70°C. (we use the -80°C)

Avoid repeated thawing and re-freezing of glycerol stocks as this can reduce the viability of the bacteria. For precious strains, storage of two stock vials is recommended.

Make a notation on the strains list and be sure to store it in the correct freezer box.

--Qiagen