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
  • 42°C 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 -20°C

  • 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 -20°C

PREPARING CELLS

  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 @ 37°C, 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 -80°C
  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°-22°C), 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 com 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

STEP 2: IMAC PURIFICATION W/ GRAVITY COLUMN

-- Main.ColinBrown - 15 Dec 2016

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Topic revision: r1 - 15 Dec 2016 - 21:27:28 - Main.ColinBrown
 
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