This is the first in a three part series on the transformation of E.coli. By the end of this you should be an expert on E.coli transformation and on which strains to choose for different applications. If you’re already an expert I hope it’ll be an enjoyable refresher for you. In either case, please comment below if you have anything to add.
Foreign DNA can be introduced into competent E.coli using electroporation or chemical transformation. Electroporation involves the application of an electric current across the cell, which is thought to create momentary “pores” in the cell membranes and force the negatively charged DNA into the cells by an electrophoresis-type effect. E.coli are made electroporation competent by thorough washing to remove all medium salts to ensure that the charge is not conducted through the medium and the electroporation is carried out at 0°C to miminize heat damage to the cells.
Chemical competence is conferred to E.coli by re-suspension in CaCl2 solution at 0°C. Under these conditions, the Ca2+ ion is thought to create pores in the membrane, assist binding of the DNA to the cell membrane and mask the negative charge on the DNA, easing it’s passage through the hydrophobic cell membrane. The DNA is forced into the cells by applying a short 42°C heat shock, which results in a thermal current that sweeps the DNA into the cells.
Electroporation is less cumbersome than chemical transformation and generally gives higher transformation efficiencies (measured in colonies formed per microgram of DNA). However, it is more expensive, requiring specialized apparatus to deliver the charge and cuvettes to transfer the charge to the cell suspension. Electroporation is sensitive to salt, so precious samples can be lost if excess salt is carried over into the cuvette.
Personally, I prefer chemical transformation. Although it takes around half-an-hour longer I find that the results are more predictable and I like the fact that a greater volume of DNA can be added if the concentration is too low – this can’t be done with electroporation because of the risk of adding too much salt to the solution. Although the commonly cited drawback of chemically transformation is its lower efficiency, with the availability of ultra-competent cells, such as Stratagene’s XL10-gold, this is no longer so much of a problem.
Like most things in this world, fluorophores are mortal, and eventually your once bright fluorescent image will inevitably fade to black. This fading or ‘photobleaching’ of fluorescent signal can make imaging difficult, especially if you are trying to take quantitative images. Read below to learn what causes photobleaching of your fluorophores and how best to […]
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