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:
- Thaw on ice 100-200 µl of chemically competent cells.
- Briefly centrifuge the ligation reaction and place on ice.
- 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.
- Incubate on ice for 30’.
- Heat shock for 45’’ in 42ºC water bath. Do not mix or shake!
- Place on ice for 1’, then add 1200 µl RT SOC or LB medium.
- Shake horizontally at 37ºC for 1 h at 200-225 RPM.
- Spread 100 µl directly onto LB plate, then pellet cells, decant supernatant, resuspend pellet in remaining 100 µl SOC and plate.
- Invert the plates and incubate at 37ºC O/N.
- Select colonies and analyze by plasmid isolation, PCR, or sequencing.
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