From 2011.igem.org

Glycerol Stocks

For the long-term storage of microbial cultures

Make sure that you do not contaminate glycerol stocks

1. Grow overnight culture (1 mL) of your cells in selective media

2. Mix 500 uL of your culture with 500 uL of 50% glycerol. Immediately place at -80 C (high concentration of glycerol is toxic to cells and prolonged exposure to it kills them at RT).

If you need to take from the stocks, do not thaw - scrape ice off of the top with a sterile loop and streak across plate.