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&mdash;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