Transformation of plasmid DNA to E. Coli

Contributed by Matt Lewis <hop2kin@yahoo.co.uk>

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.