Electrocompetent Cell Protocol
Molecular biologists routinely place fragments of DNA--plasmids--containing genes of interest into bacteria. This is a simple way to replicate and produce more of the gene and/or generate the gene's product, which could be something like insulin.
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Electroporation
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Electroporation is one method for getting the plasmid into bacteria. Bacteria and DNA are mixed in a tube and exposed to an electrical field produced by an electroporation machine. The electrical field punches small holes in the bacteria, allowing the DNA into the cells. Bacteria that take up the DNA most efficiently are electrocompetent cells.
Method
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Electrocompetent bacteria such as E. coli can be produced through a series of repeated centrifugations, first to remove their growth medium and replace it with ultra-pure water, and finally to replace the water with glycerol. In the final steps the bacteria are resuspended in a small volume of glycerol, divided into tubes, frozen in liquid nitrogen and stored long term in a -80 degree Celsius (-112 degree Fahrenheit) freezer.
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Considerations
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This protocol is usually done with large volumes that require large flasks, agitators and centrifuges. It is critical that the steps from harvest to storage all be performed at ice-cold temperatures. Ultra-pure water must be used.
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References
Resources
- Photo Credit tubes de laboratoire image by Danielle Bonardelle from Fotolia.com
