Bacterial Glycerol Stocks

1. Put 0.5ml bacterial culture in a sterile eppendorf tube.
2. Add 0.5ml of sterile 80% (v/v) glycerol soution.
3. Freeze  on dry ice or directly into –70^oc .
4. Store at –70^oC. Cells are best for about 4-6 months, but will probably work ok for a whole year. * DNA rearrangements may occur (this is especially true for bacterial strains used for protein expression). Thus, it is highly recommended to maintain DNA midi or mini preps, that will be used for future transformation in case of activity loss of the glycerol bacterial stock.

To recover the bacteria:

• Scrape the frozen surface of the culture with a sterile inoculating needle, and then immediately streak the bacteria that adhere to the needle onto the surface of an LB agar plate containing the appropriate antibiotics. Incubate the plate overnight at 37^oc.

OR

• Scrape the frozen surface of the culture with a sterile inoculating needle or tip, and immediately immerse it in 2ml growth media within a 2059 snap-cap tube. Grow the bacteria overnight in a 37^oc shaker.

Return the frozen culture to storage at -70^oc.