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Creation of Electrocompetent E. coli for Transformations

Description of protocol used to create lab stocks of electrocompetent E. coli. Below details the original protocol as well as the more commonly used one that utilizes the smaller biobottles.

Original Protocol

For use with 750ml biobottles. Makes 1L of competent cells

Materials

1 L sterile SOB, Teknova powder #S0210. Or mix:

  • 2.0% tryptone
  • 0.5% yeast extract
  • 10mM NaCl
  • 2.5mM KCl
  • 10mM MgCl2
  • 10mM MgSO4. H2O

SOC (add glucose to SOB)

  • Make 20% stock of autoclaved or filter sterilized glucose
  • Add 18mL to 1L of SOB

Sterile, preheated

  • culture tube with 2ml SOB
  • Two 2L flasks with 500mL SOC in each

Sterile, chilled

  • 2L of DI H2o
  • 200mL 10% glycerol
  • Two 750mL biobottles
  • Two 50mL conical tubes
  • XTR clinical centrifuge with 2 adapters for Biobottles and 2 adapters for 50 ml conical tubes
  • Refrigerated (or preset to 4C) microfuge
  • 2 ml Microfuge tubes (1 for last resuspension)
  • 1.5 ml Microfuge tubes (40+ in ice bucket for aliquots)
  • 1 box p200 tips, 1 box p1000 tips
  • 2 10 ml pipettes, 2 25 ml pipettes

Ice bucket

Protocol

Day 1

  1. Inoculate 2 ml SOB (or LB) with strain and grow shaking overnight at 37°C.

Day 2

  1. Inoculate 2 x 500 ml prewarmed SOC with 500 μl overnight culture and grow at 37°C until OD600 = 0.5 (approximately 2.5-3.5 hours).
  2. Cool culture quickly in an ice-water slurry.
  3. Pellet at 4000 x g, at 4°C for 15 min. Use Biobottles in the TX-450 rotor.
  4. Re-suspend each pellet in 500 ml cold dI H2O.
  5. Pellet as in #4.
  6. Re-suspend each pellet in 250 ml cold dI H2O.
  7. Pellet as in #4.
  8. Re-suspend each pellet in 20 ml cold 10% glycerol. Transfer both resuspensions to the same 50ml conical tube.
  9. Pellet as in #4, balancing with a blank tube.
  10. Re-suspend pellet in 2 ml cold 10% glycerol.
  11. Aliquot 50 μl volumes in cold microfuge tubes and store at -80ºC.

Updated Protocol

For use with 250mL biobottles. Instead of using one 750mL bottle per 500mL of culture, the culture is divided into 3 250mL biobottles. Makes 500mL of competent cells, but everything can be done in duplicate for 1L.

Materials

500mL sterile SOB (see above for recipe)

SOC - add 9mL of 20% glucose to 500mL SOB

Sterile, preheated

  • culture tube with 2ml SOB
  • One 2L flask with 500mL SOC

Sterile, chilled

  • 1L of DI H2o
  • 100mL 10% glycerol
  • Three 250mL biobottles
  • One 50mL conical tubes
  • XTR clinical centrifuge with adapters for Biobottles and 50 ml conical tubes
  • Refrigerated (or preset to 4C) microfuge
  • 1.5 ml Microfuge tubes (40+ in ice bucket for aliquots)

Ice bucket

Protocol

Day 1

  1. Inoculate 2 ml SOB with strain and grow shaking overnight at 37°C.

Day 2

  1. Inoculate 500 ml SOC with 500 μl overnight culture and grow at 37°C until OD600 = 0.5 (approximately 3-4 hours).
  2. Cool culture quickly in an ice-water slurry.
  3. Divide culture evenly into the three chilled biobottles.
  4. Pellet at 4000 x g, at 4°C for 15 min. Use Biobottles in the TX-450 rotor.
  5. Re-suspend each pellet in 165 ml cold dI H2O.
  6. Pellet as in #4.
  7. Re-suspend each pellet in 83 ml cold dI H2O.
  8. Pellet as in #4.
  9. Re-suspend each pellet in 7 ml cold 10% glycerol. Transfer all 3 resuspensions to the same 50ml conical tube.
  10. Pellet as in #4, balancing with a blank tube.
  11. Re-suspend pellet in 1 ml cold 10% glycerol.
  12. Aliquot 50 μl volumes in cold microfuge tubes and store at -80ºC.