Transformation Protocols etc

In molecular biology, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacteria must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.

Transformation is one of three processes for horizontal gene transfer, in which exogenous genetic material passes from bacterium to another, the other two being conjugation (transfer of genetic material between two bacterial cells in direct contact) and transduction (injection of foreign DNA by a bacteriophage virus into the host bacterium). In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium.

As of 2014 about 80 species of bacteria were known to be capable of transformation, about evenly divided between Gram-positive and Gram-negative bacteria; the number might be an overestimate since several of the reports are supported by single papers.

"Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because "transformation" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the process is usually called "transfection".

Protocol   need competent E Coli cells to start with here.

<ul>

<li>Thaw competent cells on ice.</li>

<li>Chill approximately 5 ng (2 μl) of the ligation mixture in a 1.5 ml microcentrifuge tube.</li>

<li>Add 50 µl of competent cells to the DNA. Mix gently by pipetting up and down or flicking the tube 4–5 times to mix the cells and DNA. Do not vortex.</li>

<li>Place the mixture on ice for 30 minutes. Do not mix.</li>

<li>Heat shock at 42°C for 30 seconds*. Do not mix.</li>

<li>Add 950 µl of room temperature media* to the tube.</li>

<li>Place tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.</li>

<li>Warm selection plates to 37°C.</li>

<li>Spread 50–100 µl of the cells and ligation mixture onto the plates.</li>

<li>Incubate overnight at 37°C.</li>

<li>* Please note: For the duration and temperature of the heat shock step as well as for the media to be used during the recovery period, please follow the recommendations provided by the competent cells’ manufacturer.</li>

</ul>

Transformation Protocols etc

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