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From 2013.igem.org

Making Glycerol Stocks
Streaking a Plate for Isolation
Pick only a single well isolated colony for making glycerol stocks.


Summary:
For the long-term storage of microbial cultures
Make sure that you do not contaminate glycerol stocks and they do not thaw.
1. Grow overnight culture (1 mL) of your cells in selective media
2. Mix 500 mL of your culture with 500 mL of 40-60% glycerol in a cryo tube (or 1.5mL tube). Immediately place at -80 C (high concentration of glycerol is toxic to cells and prolonged exposure to it kills them at RT).
3. LABEL VERY WELL and add information to drive.
See Labeling Protocol:
Freezer Stocks (Glycerol Stocks)

Plasmid Name
Backbone
Antibiotic Resistance,    Cell Type
Initials of who prepared the stock,    Date

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.

Making E-coli glycerol stock:

Material needed:

1. Growing E-coli (mid log phase)
2. 40%-60% glycerol stock (in water, autoclaved)
3. Cryo-tubes
   

To make the E-coli stock:

1. Inoculate 5 ml LB media using a single colony with appropriate antibiotics. Allow to grow to mid log phase(~12hrs).
2. In cryo-tube add 500 ul of growing E-coli.
3. Add 500 ul of 40%-60% autoclaved glycerol.
4. Mix briefly by vortexing and store at -80 C  

Growing cells from glycerol stock:
With sterile pipet tip, touch the surface of the glycerol stock (DO NOT THAW), and throw the tip in 5 ml LB media with appropriate antibiotics.