PowerPoint Presentation Recombinant DNA Bacterial TransformationStudent Instructions Making Competen

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Recombinant DNA Bacterial Transformation Student
Instructions Making Competent Cells
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Recombinant Transformation Competent Cell
Procedure
Label a sterile 1.5 ml tube with CC and your
groups name. From the Mid-Log suspension,
transfer 1 ml of culture to your sterile 1.5 ml
tube.
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Recombinant Transformation Competent Cell
Procedure
Place the tube in the microcentrifuge (MicroV
microcentrifuge, shown) and spin for 2 minutes
at 10,000 RPM.
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Recombinant Transformation Competent Cell
Procedure
Sterilely drain off the LB broth, leaving a
visible pellet of bacteria in the tube.
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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.
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Recombinant Transformation Competent Cell
Procedure
Resuspend the pelleted cell by vigorously
finger-flicking (vortexing) the tube. Place on
ice for 20 minutes.
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Recombinant Transformation Competent Cell
Procedure
After 20 minutes, place the tube in a
microcentrifuge and spin for 2 minutes at 10,000
RPM.
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Recombinant Transformation Competent Cell
Procedure
Sterilely drain off the calcium chloride, being
careful not to disturb the cell pellet.
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Recombinant Transformation Competent Cell
Procedure
Sterilely add 2 drops of fresh calcium chloride
and finger vortex to resuspend.
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Recombinant Transformation Competent Cell
Procedure
Place cells suspended in calcium chloride on ice
in the refrigerator (approx. 0oC) and store until
you are ready to use them. DO NOT FREEZE.
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Recombinant Transformation Competent Cell
Procedure
Holding the cells at 0oC 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.