Bacterial Competent Cells and Transformation

Competent Cells

For 80 ml final volume of competent cells:

  1. Inoculate 1 fresh colony into 25 ml LB. Grow at 37oC with rotation overnight

  2. Inoculate 1 L LB with the 25 ml culture. Grow at 37oC on shaker to O.D.(600) 0.4 to 0.48. Takes ~2.5 hours.

  3. Place culture on ice for 15 minutes.

  4. Spin 5 minutes in Sorvall at 6,000 rpm to pellet cells.

  5. Resuspend pellet in 300 ml ice cold RF1. Incubate on ice for 15 minutes.

  6. Spin 5 minutes in Sorvall at 6,000 rpm to pellet cells again. Aspirate off medium.

  7. Gently resuspend pellet in 80 ml ice cold RF2. Incubate on ice 15 minutes.

  8. Aliquot in 1 ml volumes and freeze at -70 to -80oC. Cells will last a long time at these temperatures.

Transformation

  1. Gently thaw cells. Don't let them get too warm. Place on ice when thawed.

  2. Incubate 50 ul of cells with your DNA on ice for 15 minutes.

  3. Heat shock at 37oC for 5 minutes. Place back on ice.

  4. Add cells to 0.5 ml LB. Incubate 20 minutes at 37oC.

  5. Plate on selective medium.


RF1 BUFFER

To make 1L: 

  • 12 g RbCl
  • 9.9 g MnCl2-4H2O
  • 30 ml 1M KOAc, pH7.5
  • 1.5 g CaCl2
  • 150 ml glycerol
  • distilled H2O to 1L
Sterile filter.

RF2 BUFFER

To make 1L: 

  • 20 ml 0.5M MOPS
  • 1.2 g RbCl
  • 11 g CaCl2
  • 150 ml glycerol
  • distilled H2O to 1L
Sterile filter.