Lactobacillus transformation (Speer 2012): Difference between revisions
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*0.5M Sucrose, 10% glycerol solution | *0.5M Sucrose, 10% glycerol solution | ||
==Procedure== | |||
#'''Grow ''L. plantarum''''' | #'''Grow ''L. plantarum''''' | ||
##Inoculate 5ml MRS medium with ''L. plantarum'' freezer stock. | ##Inoculate 5ml MRS medium with ''L. plantarum'' freezer stock. | ||
Line 46: | Line 46: | ||
##Keep on ice for 5 minutes. | ##Keep on ice for 5 minutes. | ||
##Put 1mm cuvettes on ice too. | ##Put 1mm cuvettes on ice too. | ||
#Electroporate: | #'''Electroporate:''' | ||
##Pipette the cell/DNA mixture into the cuvette | ##Pipette the cell/DNA mixture into the cuvette | ||
##Electrporate at 1200 volts | ##Electrporate at 1200 volts | ||
##Time constant should be ~5.0 | ##Time constant should be ~5.0 | ||
#Recovery: | #'''Recovery:''' | ||
##Immediately transfer the electroporated cells to 900μL of MRS medium. | ##Immediately transfer the electroporated cells to 900μL of MRS medium. | ||
##Incubate at 30°C for 2-3 hours. | ##Incubate at 30°C for 2-3 hours. | ||
#Select: | #'''Select:''' | ||
##Plate on medium with the appropriate antibiotic | ##Plate on medium with the appropriate antibiotic | ||
##Incubate at 30°C for two days. | ##Incubate at 30°C for two days. |
Revision as of 13:00, 14 September 2011
Back to Protocols
Back to Electro-transformation of Lactobacillus spp.
Back to Richard Lab:protocols
Overview
This is basically a modified version of the Mason (2005 protocol) for use with Lactobacillus plantarum and an Eppendorf 2510 electroporator.
Materials
- MRS media
- Glycine
- Milipore water
- 50mM EDTA solution
- 0.5M Sucrose, 10% glycerol solution
Procedure
- Grow L. plantarum
- Inoculate 5ml MRS medium with L. plantarum freezer stock.
- Grow overnight at 30°C without shaking.
- Make 100ml 2% glycine:
- Add 1g glycine to two flasks of 50ml MRS medium.
- Shake to dissolve.
- Add 1ml overnight culture to each flask (1:50 dlution).
- Culture for ~3 hours at 37°C with shaking.
- Wash 2x with ice-cold DI water:
- Centrifuge culture for 5min at 4000g
- Resuspend in 25ml ice-cold DI water.
- Repeat.
- Treat with ice-cold EDTA:
- Centrifuge for 5min at 4000g
- Resuspend in 10ml 50mM EDTA.
- Incubate on ice for 5 minutes
- Wash with ice-cold DI water:
- Centrifuge culture for 5min at 4000g
- Resuspend in 25ml ice-cold DI water.
- Wash 2x with Electroporation Buffer:
- Centrifuge culture for 5min at 4000g
- Resuspend in 25ml ice-cold 0.5M Sucrose, 10% glycerol.
- Repeat.
- Concentrate cells:
- Centrifuge culture for 5min at 4000g
- Resuspend in 0.8ml ice-cold 0.5M Sucrose, 10% glycerol.
- Divide cells:
- Aliquot 90μL of cell concentrate into ~20 microcentrifuge tubes.
- Keep on ice until use (within the next two hours).
- Add DNA:
- Add 10μL of plasmid DNA to the 90μL of cell concentrate.
- Keep on ice for 5 minutes.
- Put 1mm cuvettes on ice too.
- Electroporate:
- Pipette the cell/DNA mixture into the cuvette
- Electrporate at 1200 volts
- Time constant should be ~5.0
- Recovery:
- Immediately transfer the electroporated cells to 900μL of MRS medium.
- Incubate at 30°C for 2-3 hours.
- Select:
- Plate on medium with the appropriate antibiotic
- Incubate at 30°C for two days.
- Pick colony
Notes
All questions, input and feedback are welcome!
References
There will be one here soon.
Contact
- mas853@psu.edu
or instead, discuss this protocol. -->