L. acidophilus transformation
互联网
1246
Overview
Electrotransformation procedure for Lactobacillus acidophilus
Procedure
-
Prepare Electrocompetent cells
- Inoculate overnight culture at 10^6 CFU/ml in MRS containing 1% glycene
- Harvest at early-log phase (OD660 0.2-0.3)
- chill on ice for 10 minutes
- wash twice in cold washing buffer (5 mmol 1^-1 sucrose, 3 mmol 1^-1 MgCl2, pH 7.4)
- Use cells within 30 minutes
-
Electroporation
- add 1 ul of plasmid DNA to 50 ul (w/ 10^8 CFU/ml) of ice-cold cell suspension in a .2 cm cuvette
- electroporate - 12.5 kV/cm, pulse number of 10, puse interval of 500 ms, plasmid DNA concentration of 25 ng/ul
- dilute cell suspension to 1 ml in MRS broth and incubate at 37C for 3 h
- plate bacteria onto MRS agar plates with 3 ug ml^-1 chloramphenicol
- incubate under anaerobic conditions
