Transformation of plasmid DNA to competent E. Coli cells
Ver.1.0 - 7/7/2002
Material and Reagents
0.5% Yeast Extract
- 1.5 mL microfuge tubes
- Thaw competent cells on ice. 20–200µL per tube
- Add max. 20µL of a ligation reaction
- Mix very gently!
- Incubate the tubes on ice for 30 min
- Heat shock the cells for 45 sec to 2 min at 42°C
- Place the tubes immediately on ice for at least 2 min
- Add 800µL of SOC medium to each tube
- Incubate for 1 hour at 37°C and shake vigorously
- Spin down briefly and remove most supernatants
- Resuspend cell pellet with the rest SOC medium in the tube by pipetting
- Plate out the suspension on a LB agar plate containing the appropriate antibiotic.
- Incubate the plates overnight at 37°C
- I have use this protocol to transform DH5 alpha competent cells successfully
- When transforming purified plasmid into competent cells, I just add 1µL plasmid DNA solution. Then plate out only 10–20µL bacterial suspension to the plate instead of all
- Efficiency depends on ligation reaction and competent activity.
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