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Wednesday, May 4, 2011

Bacterial Transformation

1. Pipette 200ul competent cells into each of 3 ice cold Eppendorf tubes. Label the tubes Control, 1 ng, and 10 ng (1 ng is 10 -3 ug, or 10 -9 milligrams). The unknown plasmid is at a concentration of 1 ng/ul. Add 1 ng of your unknown plasmid to one tube and 10 ng to the other. Place the tubes on ice for 30 min.

2. Put the tubes at 42oC for exactly 90 seconds. Return the cells to ice for 1-2 minutes.

3. Pipette the transformation mixtures onto labeled plates containing ampicillin and spread them around using a sterilized, bent glass rod spreader. 

4. Place upside down in the 37oC incubator overnight.

5. 16 - 20 hours later, count the number of colonies on the plate with well-isolated colonies. Put parafilm around the edge of a plate and put it in a refrigerator for later use. Check the control plate to see that no colonies grew on it. 

Note: Competent Cell Preparation Procedure is given in the last post click older post to view it.

Reference

http://faculty.plattsburgh.edu/donald.slish/Transformation.html

http://www.mnstate.edu/provost/TransformationProtocol.pdf