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 750 uL of your culture with 750 uL of 40% glycerol. Immediately place at -80 C (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 - just scrape ice off of the top with a sterile loop and smear across a plate.

Double antibiotic plate

·add 20ul of the secondary antibiotic to a plate with the first antibiotic, and spread with a glass rod or inoculation loop.