Burkart:Competent Cell

RbCl Competent Cell Preparation

Materials and Equipments

50 mL LB media in a 250 mL culture flask

pre-cooled (4°C) RF1 and RF2 buffers

pre-cooled (-80°C) steriled microcentrifuge tubes

RF1 buffer

   RbCl 100mM (12g/L)

   MnCl₂·4H₂O 50mM (9.9g/L)

   Potassium Acetate 30mM (2.9g/L)

   CaCl₂·2H₂O 10mM (1.5g/L)

   Glycerol 15%(w/v)

   Adjust pH to 5.8 with acetic acid. Sterilize. Store in light-proof environment.

RF2 buffer

   RbCl 10mM (1.2g/L)

   MOPS 10mM (g/L)

   CaCl₂·2H₂O 75mM (11g/L)

   Glycerol 15%(w/v)

   Adjust pH to 6.8 with NaOH. Sterilize. Store in light-proof environment.

Protocol

1. Prepare fresh overnight 2 mL LB (with antibiotics if already containing plasmids) culture from plate colonies or frozen stocks.

2. Inoculate fresh overnight culture to 50 mL LB media in 1:250 (200μL) or 1:500 (100μL) dilution.

3. Incubate at 37°C with agitation until the Optical Density (OD₆₀₀) reaches 0.4-0.5 (about 3hr).

4. Transfer the culture into 50 mL centrifuge tube and chill the culture on ice for 10-15 min.

5. Pellet the cells by centrifugation at 1000×g for 12 min at 4°C. Drain the pelleted cells thoroughly by inverting the tubes, and use pipette to draw off recalcitrant drops of media.

6. Resuspend the cell pellet with 15 mL of pre-cooled RF1 buffer. Do it on ice and quickly.

7. Incubate the cell suspension on ice for 15 min. And pellet cells as in step 5.

8. Resuspend the cell pellet with 4 mL of pre-cooled RF2 buffer. Do it on ice and quickly.

9. Incubate the cell suspension on ice for 15 min. And quickly aliquot 50μL (or 100μL) of the cells into pre-cooled microcentrifuge tubes.

10. Immediately freeze the cells in a dry ice/isopropanol bath or liquid nitrogen, and store at -80°C.