Lactobacillus transformation (Berthier 1996): Difference between revisions
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Back to [[Protocols]]<br> | |||
Back to [[Electro-transformation of Lactobacillus spp.]] | |||
==Overview== | ==Overview== | ||
General guidelines for the electro-transformation of ''Lactobacillus sake'' as described by Berthier et al. and as used by Alegre et al. with ''Lactobacillus plantarum''. | General guidelines for the electro-transformation of ''Lactobacillus sake'' as described by Berthier et al. and as used by Alegre et al. with ''Lactobacillus plantarum''. | ||
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*Stock solution of MgCl<sub>2</sub> | *Stock solution of MgCl<sub>2</sub> | ||
*Stock Solution of Glucose | *Stock Solution of Glucose | ||
*Electroporation Buffer (0.5M Sucrose, 10% Glycerol) | |||
==Procedure== | ==Procedure== | ||
# | #Grow cells overnight in MRS Broth at 30°C. | ||
# | #Use overnight culture to inoculate 100ml MRS Broth . | ||
# | #Incubate at 30°C until OD<sub>600</sub> = 0.4-0.6. | ||
#Electroporation | #Wash 2x with wash-solution (10mM MgCl<sub>2</sub>) | ||
#Add | ##Centrifuge for 5min at 3000g. | ||
#Incubate for 2 hours at 30°C | ##Resuspend in 10ml wash-solution. | ||
#Plate | ##Repeat. | ||
#Wash 1X in Electroporation Buffer (0.5M Sucrose, 10% Glycerol) | |||
##Centrifuge for 5min at 3,000g | |||
##Resuspend in 10ml E buffer | |||
#Concentrate in Electroporation Buffer | |||
##Centrifuge for 5min at 3,000g | |||
##Resuspend in 500μL E buffer | |||
#Make 50μL aliquots and store at -80°C until use | |||
#Thaw aliquots on ice for use | |||
#Add up to 5μL plasmid DNA | |||
#Electroporate at 9000kV/cm | |||
#Recover cells by adding 500μL of MRS with 80mM MgCl<sub>2</sub> | |||
#Incubate for 2 hours at 30°C | |||
#Plate to select. | |||
==Notes== | ==Notes== | ||
*Time constants for this protocol are between 11.3 and 13.8ms using a Bio-Rad gene pulser with a pulse controller set at 25μF and 600Ω. | |||
==References== | ==References== |
Latest revision as of 18:13, 16 September 2011
Back to Protocols
Back to Electro-transformation of Lactobacillus spp.
Overview
General guidelines for the electro-transformation of Lactobacillus sake as described by Berthier et al. and as used by Alegre et al. with Lactobacillus plantarum.
Materials
- 50 ul of L.plantarum competent cells
- In vitro modified plasmid DNA
- MRS media + antibiotic
- Stock solution of MgCl2
- Stock Solution of Glucose
- Electroporation Buffer (0.5M Sucrose, 10% Glycerol)
Procedure
- Grow cells overnight in MRS Broth at 30°C.
- Use overnight culture to inoculate 100ml MRS Broth .
- Incubate at 30°C until OD600 = 0.4-0.6.
- Wash 2x with wash-solution (10mM MgCl2)
- Centrifuge for 5min at 3000g.
- Resuspend in 10ml wash-solution.
- Repeat.
- Wash 1X in Electroporation Buffer (0.5M Sucrose, 10% Glycerol)
- Centrifuge for 5min at 3,000g
- Resuspend in 10ml E buffer
- Concentrate in Electroporation Buffer
- Centrifuge for 5min at 3,000g
- Resuspend in 500μL E buffer
- Make 50μL aliquots and store at -80°C until use
- Thaw aliquots on ice for use
- Add up to 5μL plasmid DNA
- Electroporate at 9000kV/cm
- Recover cells by adding 500μL of MRS with 80mM MgCl2
- Incubate for 2 hours at 30°C
- Plate to select.
Notes
- Time constants for this protocol are between 11.3 and 13.8ms using a Bio-Rad gene pulser with a pulse controller set at 25μF and 600Ω.
References
Berthier, F., Zagorec, M., Champomier-Verge`s, M., Ehrlich, S.D. and Morel-Deville, F. (1996) Efficient transformation of Lactobacillus sake by electroporation. Microbiology 142, 1273–1279.
Alegre et al. (FEMS Microbiology Letters 241 (2004) 73–77)
Contact
- morto077@uottawa.ca
or instead, discuss this protocol. -->