Overview
Transformation of E. coli is a routine procedure in molecular biology that can be costly when using competent cells from a commercial source. Cells with a high transformation efficiency can be easily obtained in large quantities, at very low cost.
Procedure
Strike bacterial strain on LB plate containing the appropriate antibiotics if any and grow at 37C overnight
Pick single colony to inoculate 5 ml of LB with appropriate antibiotic if needed.
Grow at 37C overnight with constant agitation using a wheel drum
In the morning inoculate 250 ml of preheated LB or 2 x YT in a 1 L flask with 2.5 ml of the overnight culture.
Grow at 37C until OD600. While it is growing chill sterile milliQ water and 10% glycerol solution on ice.
Place flask in ice for 10 min. From this step on cells and solutions used should be kept on ice.
Transfer culture in 250 ml centrifuge bottle and spin 10 min at 5000 rpm at 4°C.
Discard supernatant.
Add 100 ml of cold sterile milliQ water and resuspend cells. Use pipet to break pellet if needed.
Centrifuge cells 10 min at 5000 rpm at 4C and repeat steps 9 and 10.
Resuspend pellet in 10 ml of 10% glycerol and transfer cell suspension to 50 ml sterile centrifuge tube.
Centrifuge 10 min at 5000 rpm-discard supernatant.
Repeat step 11 to 12.
Resuspend pellet in 1 volume (approx 200 µl) of 10% glycerol by gently pipetting back and forth.
Aliquot the cells in 50 or 100 µl volumes in microcentrifuge tubes and throw in liquid nitrogen before placing in deep freezer (-70C).
Use 40 µl for each transformation.
Tips
All the steps should be carried out using ice-cold solutions.
Use only ultra pure water to wash cells and to prepare 10% glycerol. The presence of impurities such as salts in the water will cause the transformation to fail.
Electrocompetent cells can be kept more than 6 months at -70C