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From 2010.igem.org

Day 1

1.Inoculate 25 mL of LB medium with the cell line you want and grow overnight with rotation at 37 degrees Celsius.

2.Autoclave the following:

    a.Eight 125 mL flasks containing 50 mL LB or SOB
    b.One liter flask containing 500 mL of millipore water
    c.One 250 mL flask containing 100 mL 10% glycerol.
 

Day 2

3.Combine the eight flasks of LB until there are 4 flasks containing 100 mL each.

4.Inoculate each flask with 5 mL of the growing cell culture and grow for three hours with rotation at 37 degrees Celsius.

5.Cool the centrifuge with the correct rotor to 4 degrees

6.At noon pour the 400 ml of culture into eight 50 ml centrifuge tubes.

7.Place the tubes on ice for 30 mins.

8.Centrifuge tubes for 10 mins at 2000g (3500 RPM)

9.Remove supernatant and gently resuspend pellets in 10ml cold sterile water.

10.Centrifuge tubes for 10 mins at 2000g

11.Remove supernatant and gently resuspend pellets in 10 ml cold sterile water.

12.Hold on ice for 30 mins.

13.Centrifuge for 10 mins at 2000g

14.Remove supernatant and gently resuspend pellets in 10 ml cold 10% glycerol.

15.Transfer to 15 ml centrifuge tubes and hold on ice for 30 mins.

16.Centrifuge for 10 mins at 2000g at 4 degrees Celsius.

17.Remove the supernatant and add 500 ul of 10% glycerol.

18.Store tubes on ice while you label 45 microcentrifuge tubes.

19.Aliquot 100 ul per tube and douse the entire tube in liquid nitrogen.

20.Place tubes in the -80 degrees Celsius freezer ASAP!