Lactobacillus transformation (Berthier 1996): Difference between revisions

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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==
#Ensure that the in vitro modification of DNA protocol for ''Lactobacillus plantarum'' has been followed.
#Grow cells overnight in MRS Broth at 30°C.
#Thaw 50 ul of competent cells on ice from aliquots in -80°C freezer.
#Use overnight culture to inoculate 100ml MRS Broth .
#Add between 1 ng and 3 ug of plasmid DNA in 5 ul of TE buffer to 50 ul of freshly prepared competent cells and transfer to prechilled cuvette for electroporation (interelectrode distance 1 mm).
#Incubate at 30°C until OD<sub>600</sub> = 0.4-0.6.
#Electroporation done at 13 Kv cm-1 (time constant: 2-4 ms), Set electroporation machine to 25μF and 200 Ω.
#Wash 2x with wash-solution (10mM MgCl<sub>2</sub>)
#Add 500 ul of MRS media containing 80mM MgCl<sub>2</sub> and 55mM Glucose
##Centrifuge for 5min at 3000g.
#Incubate for 2 hours at 30°C.
##Resuspend in 10ml wash-solution.
#Plate cells on MRS plates + antibiotic resistance.
##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==
All questions, input and feedback are welcome!
*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

  1. Grow cells overnight in MRS Broth at 30°C.
  2. Use overnight culture to inoculate 100ml MRS Broth .
  3. Incubate at 30°C until OD600 = 0.4-0.6.
  4. Wash 2x with wash-solution (10mM MgCl2)
    1. Centrifuge for 5min at 3000g.
    2. Resuspend in 10ml wash-solution.
    3. Repeat.
  5. Wash 1X in Electroporation Buffer (0.5M Sucrose, 10% Glycerol)
    1. Centrifuge for 5min at 3,000g
    2. Resuspend in 10ml E buffer
  6. Concentrate in Electroporation Buffer
    1. Centrifuge for 5min at 3,000g
    2. Resuspend in 500μL E buffer
  7. Make 50μL aliquots and store at -80°C until use
  8. Thaw aliquots on ice for use
  9. Add up to 5μL plasmid DNA
  10. Electroporate at 9000kV/cm
  11. Recover cells by adding 500μL of MRS with 80mM MgCl2
  12. Incubate for 2 hours at 30°C
  13. 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. -->

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