Quint Lab:e.coli transformation chemical

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chemical transformation of e.coli

Before starting:

  • Equilibrate water bath to 42ºC
  • Spread x-gal and IPTG onto LB agar plates with antibiotic, if desired
  • Warm the plates to 37ºC for 30’
  • Make sure to use competent cell with transformation efficiency of 107 cfu/µg DNA for ligations with single digest sticky ends and 108 cfu/µg DNA for ligations double digest sticky ends/blunt ends

Procedure:

  1. Thaw on ice 100-200 µl of chemically competent cells.
  2. Briefly centrifuge the ligation reaction and place on ice.
  3. Pipet 1-5 µl of the ligation reaction directly (if possible) into the competent cells (100-200 µl) and mix by tapping gently. Do not mix by pipetting! Store the remaining ligation reaction at –20ºC.
  4. Incubate on ice for 30’.
  5. Heat shock for 45’’ in 42ºC water bath. Do not mix or shake!
  6. Place on ice for 1’, then add 1200 µl RT SOC or LB medium.
  7. Shake horizontally at 37ºC for 1 h at 200-225 RPM.
  8. Spread 100 µl directly onto LB plate, then pellet cells, decant supernatant, resuspend pellet in remaining 100 µl SOC and plate.
  9. Invert the plates and incubate at 37ºC O/N.
  10. Select colonies and analyze by plasmid isolation, PCR, or sequencing.
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