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.