Team:RMIT Australia/Notebook/Transformation


Transformation of plasmid DNA into Ca-competent cells

1. Pre-incubate LB-agar plates at 37°C for 2h before transformation.
2. Thaw Ca-competent cells on ice (4ºC).
3. Add plasmid DNA 3μl to each tube of 50μl of prepared competent cells. Mix the contents of the tubes by gently swirling and immediately store the tubes on ice for 30mins.
4. Transfer the tubes to a rack placed in a preheated water bath (42ºC) and incubate for 42secs. Warning! Do not shake the tubes.
5. Transfer the tubes to ice (4ºC). Incubate on ice for 2 min.
6. Add 800μl SOC medium to each tube. Incubate the tubes at 30ºC at 250rpm for 45min.
7. Transfer 50μl of the transformed cells onto agar plates and spread the cell suspension thoroughly on the surface using a sterile rod.
8. Transfer plates to the incubator. Incubate (agar side up) at 37ºC for ~16h.