Griffitts:Electrocompetent Cells

Procedure

Note: This should be done immediately prior to transformation; cells are no longer competent following flash freezing so they cannot be stored

Using a Sorvall centrifuge

• Grow recipient strain at 30°C (for S. meliloti) or 37°C (for E. coli) overnight in 25 mL LB broth
• Inoculate 100 mL LB broth with 250 μL of the overnight culture
• Shake at 30°C (for S. meliloti) or 37°C (for E. coli) until OD₆₀₀ = 0.5
  • After this point, keep all cultures and liquids on ice
• Spin at 8000 rpm for 10 minutes in cold SS34 rotor
  • The Sorvall Centrifuge is found in the Harker/Breakwell lab (747 WIDB)
  • Divide the culture into 2 tubes with ~40 mL each
• Remove all supernatant
• Gently resuspend with 15 mL cold 10% glycerol
• Spin at 8000 rpm for 10 minutes
• Remove all supernatant
• Gently resuspend with 15 mL cold 10% glycerol
• Spin at 10,000 rpm for 10 minutes
• Remove all supernatant (the pellet is especially fragile at this point)
• Gently resuspend with ~200 μL cold 10% glycerol and combine
• Immediately place the cells on ice

Using an Eppendorf microcentrifuge

Note: This has only been tried once—so it is not guaranteed and may be subject to revision

• Grow recipient strain at 30°C (for S. meliloti) or 37°C (for E. coli) overnight in 4 mL LB broth
• Inoculate 4 mL LB broth with 100 μL of the overnight culture
• Shake at 30°C (for S. meliloti) or 37°C (for E. coli) until OD₆₀₀ = 0.5
  • After this point, keep all cultures and liquids on ice
• Divide culture into four 1.5-mL Eppendorf microfuge tubes (~1 mL culture per tube)
• Spin at 13,2000 rpm for 30 seconds
• Dump and tap
• Gently resuspend with 0.5 mL cold 10% glycerol
• Spin at 13,2000 rpm for 30 seconds
• Dump and tap
• Gently resuspend with 0.5 mL cold 10% glycerol
• Spin at 13,2000 rpm for 30 seconds
• Dump and tap (the pellet is especially fragile at this point)
• Gently resuspend in the liquid that remains in the tube and combine
• Immediately place the cells on ice

Transformation

• Add 1.5 μL DNA solution to 50 μL electrocompetent cells
• Transfer electrocompetent cells with added DNA into a chilled electroporation cuvette
• Shock
  • There are electroporators in the MMBIO 460 lab (794 WIDB) and in Dr. Erickson's lab (844 WIDB)
  • Use the EC1 setting
• Immediately add 400 μL LB broth and pipet up and down
• Transfer to an Eppendorf tube
• Place on shaker at 30°C for 1 hour
• Plate 10 μL onto selective medium