UCL London/Protocol/Transformation

Transformation


 * Materials:


 * Plasmids DNA
 * Competent E.coli
 * NZY Medium
 * LB buffer
 * Ampicllin, Kanamycin, Tetracycline
 * Agar Plates
 * Eppendorf


 * Method 1:


 * 1) Add 5µL ice-cold plasmid DNA into the competent E.coli on ice.
 * 2) Incubate on ice for 30 min. (minimum) Incubate at 42°C for 45 sec.
 * 3) Incubate on ice for 2 min. (minimum)
 * 4) Add 300µL of NZYX medium.
 * 5) Incubate with shaking at 37°C for 1 hr.
 * 6) Spin the incubated E.coli suspension for 3min at maximum speed. (Optional)
 * 7) Discard about half of the liquid, and then re-suspend the E.coli solution. (Optional)
 * 8) Spread the suspension onto selective agar plates.
 * 9) Incubate at 37°C overnight.
 * 10) Pick a colony into 2ml selective LB (Add 100µL antibiotic to 50mL LB medium). Incubate 37°C in shaker overnight


 * Method 2:


 * 1) Using the puch tool, puch out the appropriate DNA. Clean the tool between punches.
 * 2) Soak the spots in 5µL of TE that has been warmed to 50ºC for 20 minutes. At this time take the competent cells from the -80º freezer and start defrosting on ice.
 * 3) Add 2µL of DNA in TE to 50µL of competent cells (TOP10)
 * 4) Allow the DNA and competent cells to sit on ice for 30 minutes
 * 5) Heat shock at 42ºC for 60 sec in water bath.
 * 6) Recover on ice for 5 min.
 * 7) Add 200 µL SOC media.
 * 8) Incubate at 37ºC for 2 hr while the tubes are rotating.
 * 9) Centrifuge and leave about 100 µL liquid; hence, resuspend the E.coli suspension.
 * 10) Plate 250µL on an LB plate with the appropriate antibiotic.