L.acidophilustransformation

Overview

Electrotransformation procedure for Lactobacillus acidophilus

Procedure

1. Prepare Electrocompetent cells

1. Inoculate overnight culture at 10^6 CFU/ml in MRS containing 1% glycene

2. Harvest at early-log phase (OD660 0.2-0.3)

3. chill on ice for 10 minutes

4. wash twice in cold washing buffer (5 mmol 1^-1 sucrose, 3 mmol 1^-1 MgCl2, pH 7.4)

5. Use cells within 30 minutes

2. Electroporation

1. add 1 ul of plasmid DNA to 50 ul (w/ 10^8 CFU/ml) of ice-cold cell suspension in a .2 cm cuvette

2. electroporate - 12.5 kV/cm, pulse number of 10, puse interval of 500 ms, plasmid DNA concentration of 25 ng/ul

3. dilute cell suspension to 1 ml in MRS broth and incubate at 37C for 3 h

4. plate bacteria onto MRS agar plates with 3 ug ml^-1 chloramphenicol

5. incubate under anaerobic conditions