Transformation of plasmid DNA to E. Coli¶
Contributed by Matt Lewis <firstname.lastname@example.org>
Transformation of plasmid DNA to competent E. Coli cells
- Thaw competent cells on ice. 20–200µL per tube
- Add max. 20µL of a ligation reaction and mix _very_ gently
When transforming purified plasmid into competent cells add just 1ul plasmid DNA solution.
- Incubate the tubes on ice for 30 min
- Heat shock the cells at 42’C for 45 sec to 2 mins
- Place the tubes immediately on ice for a further 2 minutes
- Add 800µL SOC medium to each tube
- Incubate for 1 hour at 37°C shaking vigorously
- Spin down at 1200rpm for 5 minutes and remove supernatant
- Resuspend cell pellet in 200ul SOC medium by pipetting up and down
- Plate out the suspension on a LB agar plate containing the appropriate antibiotic.
When transforming purified plasmid into competent cells plate out only 10–20µL bacterial suspension to the plate instead of all.
- Incubate the plates overnight at 37°C
This method is based, with permission, on an original protocol available here.