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From 2012.igem.org

I. Check overnight cultures for growth

• As of ~2:30 PM when I got in, both cultures were cloudy corresponding to saturation

II. Make 60% glycerol stock from 100% stock

• Dispense 1.6 mL of sterile dH₂O into a 15 mL conical tube
• Add 2.4 mL of sterile 100% glycerol, making sure to wait to make sure the viscous fluid is completely dispensed
• Mix thoroughly by vortex

III. Make 15% Glycerol Stocks (6 total) from Successful Overnight Cultures

• Transfer 600 uL of each liquid culture to each microcentrifuge tube (x3 each)

• ECNR1
• ECNR2

• Add 200 uL of 60% glycerol to each tube (x3 each)
• Vortex to mix thoroughly
• Store tubes in the -80 ^oC freezer
• Discard any remaining liquid culture from above cell line