Lactobacillus transformation (Kim 2005): Difference between revisions
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==Procedure== | ==Procedure== | ||
#Prepare Electrocompetent cells | #'''Prepare Electrocompetent cells:''' | ||
## | ##Use overnight culture (10<sup>6</sup> CFU/mL) to inoculate 100mL MRS containing 1% glycine. | ||
## | ##Grow until OD<sub>660</sub> = 0.2-0.3. | ||
##chill on ice for 10 | ##Place culture into two 50mL centrifuge tubes and chill on ice for 10 mins. | ||
## | ##Wash 2X with cold washing buffer (5 mM NaH<sub>2</sub>PO<sub>4</sub>, 1mM MgCl<sub>2</sub>, pH 7.4): | ||
###Centrifuge for 5min at 4000g. | |||
###Resuspend in 10ml washing-buffer. | |||
###Repeat. | |||
##Use cells within 30 minutes | ##Use cells within 30 minutes | ||
#Electroporation | #'''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 | ##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 | ##electroporate - 12.5 kV/cm, pulse number of 10, puse interval of 500 ms, plasmid DNA concentration of 25 ng/ul | ||
Line 20: | Line 23: | ||
##plate bacteria onto MRS agar plates with 3 ug ml^-1 chloramphenicol | ##plate bacteria onto MRS agar plates with 3 ug ml^-1 chloramphenicol | ||
##incubate under anaerobic conditions | ##incubate under anaerobic conditions | ||
==Notes== | ==Notes== |
Revision as of 18:25, 16 September 2011
back to protocols | ||
Back to Protocols
Back to Electro-transformation of Lactobacillus spp.
Overview
Electrotransformation procedure for Lactobacillus acidophilus, Lactobacillus brevis, and Lactobacillus helveticus.
Procedure
- 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 10ml washing-buffer.
- Repeat.
- 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
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.