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Chemical transformation

Overview

Sterile technique must be strictly observed – work next to an open flame. All centrifuge bottles, tubes, solutions, pipets, etc. must be sterile.

Materials

  • DH5alpha / Top10 of E. coli cells – -80C fridge
  • 50 mM CaCl2 (ice cold)
  • 50 mM CaCl2 with 100% glycerol (86% of CaCl2 and 14% of glycerol)
    • If anhydrous – 5.55 g for 1 L water
    • If monohydrate – 6.45g for 1 L water
    • If dihydrate – 7.35g for 1 L water
  • LB media
  • Spectrophotometer
  • 37°C shaking incubator (200 rpm)
  • Ice bucket

Procedure

Chemically competent cell preparation

  1. Inoculate 1 ml of LB with cells from a frozen glycerol stock and incubate overnight at 37ºC.
  2. Transfer the entire 1 ml into a 1 L Erlenmeyer flask containing 500 ml LB and incubate at 37ºC.
  3. Monitor growth starting at about 2 hours by measuring OD of an aliquot. Let cells grow until the OD550 = 0.45 to 0.6. This should take approximately 4 hours.
  4. Transfer the culture to 2 x 500 ml centrifuge bottles and chill on ice to 4ºC.
  5. Centrifuge at 6,000 g for 5 min at 4ºC (cool ahead of time).
  6. Pour off supernatant and pipette off remaining supernatant.
  7. Resuspend each pellet in 125 ml ice cold 50mM CaCl2. Combine into a single 500 ml centrifuge bottle.
  8. Centrifuge at 6,000 g for 5 min at 4ºC.
  9. Pour off supernatant and resuspend in 21.5 ml ice cold CaCl2:glycerol solution
  10. Prepare 500 µl aliquots in sterile microfuge tubes and snap freeze in liquid nitrogen
  11. Store in -80ºC freezer. Use within 6 months if possible

Transformation

  1. Prewarm water bath to 42C.
  2. Prewarm SOC recovery medium to 37C.
  3. Thaw a tube of electrocompetent cells immerse in ice for 2-5 min and gently flick to resuspend them.
    • Competent cells are very fragile!
  4. Add 50 uL cells to the tube.
  5. Add 1 uL (1 pg-100 ng) of plasmid DNA to cells and mix without vortexing.
  6. Incubate on ice for 10 min
  7. Heat shock tubes in water bath (42C) for 45 sec.
  8. Place on ice for 3 min.
  9. Add 900 uL prewarmed SOC.
  10. Proceed to plating.

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