Preparing Chemically Competent Cells using the Inoue Method

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:

• SOB Medium (20mM MgSO4)

• SOC Medium

• 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:

• • Filter sterilize and store 40mL aliquots in 50mL Falcon Tubes at -20°C

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 @ 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 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 4°C
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 4°C
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 -80°C until needed

Preparing Chemically Competent Cells using the Inoue Method

• Floor Centrifuge
• Sterile 500mL Centrifuge Bottles
• Shaking Incubator or Shaking Platform and 1L flask clamps

• Sterile 1L Flasks

• Liquid Nitrogen
• Polypropylene Microcentrifuge Tubes

• SOC Medium

• Transformation Buffer:

Preparing Chemically Competent Cells using the Inoue Method

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:

• SOB Medium (20mM MgSO4)

• SOC Medium

• 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