Eletrocompetent E. Coli cells

(Based on the protocol from Bio-Rad Laboratories)

  1. Inoculate 500ml Terrific Broth with 1/100 volume of fresh overnight culture
  2. Grow the cells at 37C shaker at 300 rpms to an OD600 of ~2.4
  3. Chill cells on ice for 20 minutes. From this point on everything possible is done in the cold room and on ice.
  4. Transfer cells to iced centrifuge bottles and spin in fixed angle rotor 5k rpm 15 min at 4C
  5. Pour off all supernatant and resuspend in 500ml iced 10% glycerol. Spin 5k rpm 15 min at 4C
  6. Resuspend in 250ml iced 10% glycerol. Spin 5k rpm 15 min at 4C
  7. Resuspend in 20ml iced 10% glycerol. Count 5ul in a Coulter Counter to determine the final volume. You want ~ 1-3x1010 cell/ml. Transfer to a 30mL sterile Oakridge rube and spin at 5k rpm 15min at 4C
  8. Resuspend cells in iced 10% glycerol to calculated final volume
  9. Using a 8 channel repetitive pipettor, dispense 40ul into strips of 8 – 0.2ml tubes
  10. Freeze in liquid nitrogen and store at –70C. Good for at least 6 months

Transforming Electrocompetent E. coli Cells

  1. On ice, add 1-2ul of ligation reaction to 40ul of electrocompetent cells, mix gently and incubate for 1 min.
  2. Transfer cells/DNA to iced 0.2cm electroporation cuvette, tap cells to the base of the cuvette
  3. Set Micropulser to E2 (setting for 0.2cm cuvettes), slide cuvette in to chamber and pulse once.
  4. IMMEDIATELY add 1ml SOC media to cuvette and mix by pipeting
  5. Transfer cells to 1.5ml centrifuge tube, spin max for 1 min, and resuspend in 100ul TE.
  6. Spread transformants on selective plate and incubate over night at 37C

Determining Efficiency of Competent Cells

  1. Transform one aliquot of competent bacteria with 2ul of 10ng/ml pUC19 plasmid.
  2. After adding SOC, plate 100ul and 10ul on TYE + Amp plates
  3. Grow over night at 37C
  4. Count colonies

Calculation 1: amount of DNA

0.002ml x 10ng/ml x 1ug/1000ng = 2x10-5 ug

Calculation 2: efficiency

Z colonies / 10ul (from transformation mix) x 1000ul (total transformation mix) x 1/(2x10-5ug)