How to practically carry out Bacterial Transformation

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jackthomas
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How to practically carry out Bacterial Transformation

Post by jackthomas » Sun Feb 08, 2009 3:31 pm

Bacterial transformation is routine work in all molecular biology laboratories as part of recombinant DNA experiment or gene cloning. In rDNA experiments or gene cloning, we prepare recombinant DNA or the gene or plasmid to be cloned, which has to be transferred to a host cell so that the DNA will multiply inside the bacterial cell. Transfer of the plasmid or the rDNA is carried out by bacterial transformation.

The first step is to select a suitable host cell such as a suitable strain of e.coli like DH5 , a common strain available in all molecular biology laboratories, which can take foreign DNA easily. For this we have to treat the grown bacterial cultures at its log phase of growth, with CaCl2. Centrifuge the cells growing at the log phase under low rpm (3,000-5,000 for 10 minutes) at 4°C and collect the cells. Suspend the cells in chilled CaCl2 of 0.1 M. The cells in calcium chloride are able to accept the small DNA molecules. These cells in CaCl2 can be stored for a long time under low temperatures such as -20 or -70°C.

Sudden exposure of this cell to the room temperature or higher can force the cell to take the DNA from outside. Take the stored competent cells, which are in the frozen condition and add the DNA sample to these cells and expose them to a higher temperature, at 42°C for two to three minutes. Some of these cells take the DNA from outside and will be transformed by intercalating with its genome. These cultures can be plated on a selection agar plate and the transformed colonies can be selected against the untransformed ones.

This transformation is extensively used in genetic engineering experiments. Any gene or DNA, before transferring into an organism, can be tested in a selected host by this transformation method. New promoters can be checked for their strength of expression. Commercially-useful enzymes and therapeutic proteins can be prepared in industrial scales. In short, any genetic engineering or gene cloning cannot be accomplished without bacterial transformation.

Article By:
bio-genetix.blogspot.com

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