Preparing Chemically Competent Cells using the Inoue Method

Adapted from Molecular Cloning: A Laboratory Manual 3rd Ed., Sambrook and Russell (2001)

SUPPLIES:

Equipment:

  • Floor Centrifuge
  • Sterile 500mL Centrifuge Bottles
  • Shaking Incubator or Shaking Platform and 1L flask clamps
  • 42C Heating Bath or Block
  • Sterile 1L Flasks

Consumables:

  • Liquid Nitrogen
  • Polypropylene Microcentrifuge Tubes

Buffers and Solutions:

  • 0.5 M PIPES (pH 6.7):
    • Dissolve 15.1g PIPES in 80mL MilliQ / UltraPure H2O, adjust pH to 6.7 w/ 5M KOH (measure 1mL aliquots in a 15mL falcon tube to avoid contaminating stock solution). Adjust volume to 100mL and filter sterilize, divide into 10mL aliquots in 15mL Falcon tubes, and store at -20C

  • Transformation Buffer:
Reagent Amount / L Final Conc.
MnCl2 • 4H2O 10.88 g 55 mM
CaCl2 • 2H2O 2.20 g 15 mM
KCl 18.65g 250 mM
PIPES (0.5 M, pH 6.7) 20 mL 10 mM
H2O to 1 L  
    • Filter sterilize and store 40mL aliquots in 50mL Falcon Tubes at -20C

PROTOCOL

  1. ) First thing in the morning, seed a starter culture in 25mL LB in a 250mL flask with a single colony from a freshly grown overnight plate. Grow this culture 6-8 hours @ 37C, 300 rpm.
    • Alternatively, you can prepare frozen starter cultures by growing a 50mL culture overnight, dividing into aliquots, adding 0.25 volumes 80% Glycerol, and freezing at -80C
  2. ) At the end of the day (~6pm) inoculate 3 1L flasks containing 250mL SOB with 10mL, 4mL, and 2mL of fresh or frozen starter culture. Incubate these on a shaking platform at room temp (~18-22C), shaking at 200-250 rpm.
    • The point of this is to make sure at least one of these cultures will reach the right OD when you come in the next morning.
  3. ) The following morning, read the OD600 of all three cultures. Measure every 45min until one of the cultures reaches an OD600 of 0.55; keep this culture and discard the other two.
  4. ) Place the culture flask in an ice bath for ~10min.
  5. ) Harvest cells by centrifuging at 2500xg for 10min at 4C
  6. ) Pour off supernatant and invert the centrifuge bottle on a paper towel for two min; remove any remaining drops of medium with a pipette
  7. ) Resuspend cells by gently swirling in 0.32x the original culture volume ice-cold transformation buffer (e.g. 80mL buffer if you started w/ a 250mL culture)
  8. ) Harvest cells by centrifuging at 2500xg for 10min at 4C
  9. ) Pour off remaining medium and remove any remaining drops
  10. ) Resuspend cells in 0.08x original culture volume ice-cold transformation buffer (e.g. 20mL buffer if you started w/ a 250mL culture)
  11. ) Add DMSO to a final concentration of 7% (so add 1.5mL if your resuspended cell solution is 20mL)
  12. ) Dispense 50uL aliquots into pre-chilled centrifuge tubes, then close tubes tightly and snap-freeze in liquid nitrogen
  13. ) Store cells at -80C until needed

-- Main.ColinBrown - 15 Dec 2016

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Topic revision: r2 - 23 Feb 2017 - 21:37:51 - Main.ColinBrown
 
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