Difference between revisions of "Team:Paris Bettencourt/Protocols/Electrocompetent Cells Preparation for Lactobacillus Plantarum"

 
 
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     <td><b>Electrocompetent Cells Preparation Protocol for Lactobacillus Plantarum</b>
 
     <td><b>Electrocompetent Cells Preparation Protocol for Lactobacillus Plantarum</b>

Latest revision as of 17:34, 14 August 2015

Electrocompetent Cells Preparation Protocol for Lactobacillus Plantarum
  • Inoculate 5ml MRS medium with L. plantarum freezer stock.
  • Grow overnight at 30°C without shaking.
  • Add 1.25g 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.
  • Centrifuge culture for 5min at 4000g
  • Resuspend in 25ml ice-cold DI water.
  • Repeat it.
  • Centrifuge for 5min at 4000g
  • Resuspend in 5ml 50mM EDTA.
  • Incubate on ice for 5 minutes
  • Add 25ml ice-cold DI water.
  • Centrifuge culture for 5min at 4000g
  • Resuspend in 25ml ice-cold DI water.
  • Centrifuge culture for 5min at 4000g
  • Resuspend in 25ml ice-cold 0.5M Sucrose, 10% glycerol.
  • Repeat it.
  • Centrifuge culture for 5min at 4000g
  • Resuspend in 0.8ml ice-cold 0.5M Sucrose, 10% glycerol.
  • Aliquot 90μL of cell concentrate into ~20 microcentrifuge tubes.
  • Keep on ice until use (within the next two hours).
  • Add 10μL of plasmid DNA to the 90μL of cell concentrate.
  • Keep on ice for 5 minutes.
  • Put 1mm cuvettes on ice too.
  • Pipette the cell/DNA mixture into the cuvette
  • Electrporate at 1200 volts
  • Time constant should be ~5.0
  • Immediately transfer the electroporated cells to 900μL of MRS medium.
  • Incubate at 30°C for 2-3 hours.
  • Plate on medium with the appropriate antibiotic
  • Incubate at 30°C for two days.
  • Pick colony