Week Seven

 This week marks the start of the transformation process of Deinococcus caeni. Before we are able to perform the transformation procedures there are a number of things that must be prepared. Transformation occurs naturally in the environment under selective pressures that cannot accurately replicated, thus the bacterial cells ability to be transformed must be included. We call these included cultures competent cells. Firstly, competent cells of the bacteria in focus must be prepared. The competent cell are chemically prepared in the lab. Sodium chloride, or salt, is used to neutralize the negative charge of DNA and the phospholipid bilayer in the cell membrane, promoting the DNA to move closer to the cell. The cells are stored in the –80 degrees Celsius freezer to maintain the permeability of the cell membrane while the glycerol prevents the cells from freezing and lysing.  

 

Materials: 

-TGY broth 

-Glycerol 60% 

-DI water 

-D. Caeni culture 

-Microcentrifuge tubes  

-Microcentrifuge 

-Cryovials 2ml 

-1M Sodium chloride  

Protocol: 

1. Pipette 1ml of the D. caeni bacterial culture broth into a sterile microcentrifuge tube. 

2. Centrifuge for 1 minute using the centrifuge at 12,000 rpm, after which dispose of the supernatant. 

3. Resuspend pellet in the following mixture: 

-1290 ul TGY broth 

-51.6 ul 1M Sodium chloride 

-335.1 ul DI water 

-323 ul Glycerol 60% 

4. Mix well and place the solution in a 2 ml cryovial. 

5. Store in the –80 degrees Celsius freezer. 

 

Cells that are not made competent will not uptake genetic information in their environment. Thus, cells that are expected to be transformed will not grow on selective pressure plates. A false negative result may be suggested from a lack of bacterial growth. It is essential to perform this procedure before new DNA material is introduced to the bacteria to ensure that a lack of growth on selective plates is a result of the cells incapacity to be transformed or other experimental errors.  

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