Burkart:Competent Cell

Burkart Lab

Home        Protocols        Equipment        Lab Meeting        Orders        Calendar        Links        Contact us        Publications       

RbCl Competent Cell (E. coli) Preparation

Introduction

Escherichia coli cells are grown to log phase. Exposure to calcium ions renders the cells able to take up DNA, or competent. Cells are treated with a hypotonic solution containing RbCl expand. As a result, the expulsion of membrane proteins allows negatively charged DNA to bind. The mixture of DNA and cells is then heat shocked or electroporated, which allows the DNA to efficiently enter the cells.

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 MnCl2·4H2O Potassium Acetate CaCl2·2H2O Glycerol	100mM (12g/L) 50mM (9.9g/L) 30mM (2.9g/L) 10mM (1.5g/L) 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.

Note

1. The growiing phase (O.D.₆₀₀) of 50 mL culture is critically important to the quality of competent cells.

2. From the step 4 on, keep every process on ice and do it quickly. High temperature and long processing time will decrease the cell quality.

3. For saving time, ask somebody to help you opening, recapping and freezing the microcentrifuge tube while aliquoting the cell suspensions.

4. Test your competent cell efficiency (c.f.u.) with 1 pg of plamid DNA by conventional Heat-Shock transformation. The effeciency of a good-quality competent cell (For DH5α) should be higher than 10⁷ c.f.u.