From 2014.igem.org

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4^th June

• DAY THREE : Grow cells until A600: 0.5 – 0.7
  
  
  Subsequent steps should be carried out in the cold room on ice :
  Put flask on ice for 10 minutes, then spin cells down at 2500xg (3350 RPM) in JLA 8.1 rotor. Pour off supernatant. Resuspend gently in 80 mL – I first resuspend in 25 mL TB, then add remaining 295 mL. Leave on ice for 10 minutes; spin down again at 2500 xg for 10 minutes. Resuspend cells in 20 mL TB, add 1.4 mL of DMSO dropwise while gently shaking [Final DMSO concentration is 7%]. Aliquot in 100 μL aliquots (you’ll need about 450 pre-chilled 0.5 mL tubes). Flash freeze in liquid nitrogen. Store at -80°C (the lower the rack the better).