Lactobacillus transformation (Kim 2005): Difference between revisions

#'''Prepare Electrocompetent cells:'''

##Use overnight culture (10⁶ CFU/mL)  to inoculate 100mL MRS containing 1% glycine.

##Grow until OD₆₆₀ = 0.2-0.3.  

##Place culture into two 50mL centrifuge tubes and chill on ice for 10 mins.

##Wash 2X with cold washing buffer (5 mM NaH₂PO₄, 1mM MgCl₂, pH 7.4):

###Centrifuge for 5min at 4000g.

###Resuspend in unspecified amount of washing-buffer.

###Repeat.

##Concentrate in electroporation-buffer (1mM sucrose, 3mM MgCl₂, pH 7.4):

###Centrifuge for 5min at 4000g

###Resuspend in unsepcified amount of electroportion buffer.

##Keep on ice and transform 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