1. Recombinant DNA Bacterial Transformation Student Instructions Making Competent Cells

2. Recombinant Transformation Competent Cell Procedure Label a sterile 1.5 ml tube with “CC” and your group’s name. From the Mid-Log suspension, transfer 1 ml of culture to your sterile 1.5 ml tube.

3. Recombinant Transformation Competent Cell Procedure Place the tube in the microcentrifuge (MicroV microcentrifuge, shown) and spin for 2 minutes at 10,000 RPM.

4. Recombinant Transformation Competent Cell Procedure Sterilely drain off the LB broth, leaving a visible pellet of bacteria in the tube.

5. Recombinant Transformation Competent Cell Procedure Use a sterile plastic pipette to transfer 0.5 ml (10 drops) of calcium chloride to the tube containing the cells.

6. Recombinant Transformation Competent Cell Procedure Resuspend the pelleted cell by vigorously finger- flicking (vortexing) the tube. Place on ice for 20 minutes.

7. Recombinant Transformation Competent Cell Procedure After 20 minutes, place the tube in a microcentrifuge and spin for 2 minutes at 10,000 RPM.

8. Recombinant Transformation Competent Cell Procedure Sterilely drain off the calcium chloride, being careful not to disturb the cell pellet.

9. Recombinant Transformation Competent Cell Procedure Sterilely add 2 drops of fresh calcium chloride and finger vortex to resuspend.

10. Recombinant Transformation Competent Cell Procedure Place cells suspended in calcium chloride on ice in the refrigerator (approx. 0 o C) and store until you are ready to use them. DO NOT FREEZE.

11. Recombinant Transformation Competent Cell Procedure Holding the cells at 0 o C for 24 hours increases the cell competency. Cells can be held on “wet ice” (combination of melted and solid ice) for several days while you make the recombinant constructs.