Recombinant DNA Bacterial Transformation Student Instructions Making Competent Cells.

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Recombinant DNA Bacterial Transformation Student Instructions Making Competent Cells

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.

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

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

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.

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

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

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

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

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.

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.