Lactobacillus transformation (Kim 2005)

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(New page: ==Overview== Electrotransformation procedure for ''Lactobacillus acidophilus'' ==Procedure== #Prepare Electrocompetent cells ##Inoculate overnight culture at 10^6 CFU/ml in MRS containi...)
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==Notes==
==Notes==
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
 +
'''*[[User:Derek Ju|Derek Ju]] 04:44, 29 October 2008 (EDT)''':used plasmid pNZ123, works with L. acidophilus strains 43121, 4356, NCFM, 30SC, A4, 107A, GP4A; L. helveticus KU107; L. brevis 3102   
'''*[[User:Derek Ju|Derek Ju]] 04:44, 29 October 2008 (EDT)''':used plasmid pNZ123, works with L. acidophilus strains 43121, 4356, NCFM, 30SC, A4, 107A, GP4A; L. helveticus KU107; L. brevis 3102   

Revision as of 03:45, 29 October 2008

Contents

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


Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

*Derek Ju 04:44, 29 October 2008 (EDT):used plasmid pNZ123, works with L. acidophilus strains 43121, 4356, NCFM, 30SC, A4, 107A, GP4A; L. helveticus KU107; L. brevis 3102

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers and books Kim et al (Journal of App. Microbio. 2005, 99, 167-174)

Contact

  • Derek Ju (derekju [at] mit.edu)

or instead, discuss this protocol.

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