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Team:Cambridge/Protocols/Transformation of E.Coli
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| - | <sup>[http://www.ias.ac.in/currsci/dec102002/1376.pdf]</sup> | + | <sup>[http://www.ias.ac.in/currsci/dec102002/1376.pdf]</sup> Mechanism of artificial transformation of E. coli with plasmid DNA – Clues from the influence of ethanol |
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Revision as of 23:31, 21 July 2011
Transformation of E.Coli
A simple method of transforming competent E.coli cells with your DNA of choice.
Preparation
Often you will have to do some steps well in advance of carrying out a protocol. In this case you must take liquid broth to be autoclaved in good time. You should also turn on the water bath and incubator so they are at the required temperature when you need them, but make sure you turn them off after too.
Theory
Artificial transformation is a process whereby E.coli are made competent and take up DNA from their surroundings. The main steps are chilling the cells to 0ºc with CaCl2 solution, adding the DNA and then heat shocking the bacteria for a short period of time.
One hypothesis for artificial transformation is that the divalent cation provided by the chilled CaCl2 solution which is used in creating competent cells promotes an interaction between DNA and lipopolysaccharides which are also negatively charge. Lipopolysaccharides occur in higher densities near the parts of the outer membrane of Gram negative bacteria that are in close association with the inner membrane (zones of adhesion).
It is believed that prior to incubation DNA may interact with lipopolysaccharides and then cross the 'least barrier path' at the zones of adhesion described above.
Practice
Take care not to disturb the competent E.coli, in other words do not vortex them or place them on ice.
To a clean Eppendorf tube add
| Reagent | Quantity (μl) |
| Competent E.coli cells | 50 |
| DNA | 10 |
Incubate the cells with the DNA at 42ºC for 1 minute.
Add 250μl of liquid broth (which should have already been autoclaved)
Incubate at 37ºc for 1 hour.
Plate 10μl on agar containing an antibiotic (the antibiotic for which resistance is included in the DNA being taken up by the cells) and also plate 100μl on another identical plate. This allows for possibly overcrowding as the next step after this protocol usually requires a single colony to be selected.
Incubate overnight at 37ºC
Always keep agar plates upside down so that drips of condensation and falling debris does not contaminate them.
Safety
All equipment (including gloves) that may have come into contact with the bacteria must be autoclaved for decontamination purposes.
References
[http://www.ias.ac.in/currsci/dec102002/1376.pdf] Mechanism of artificial transformation of E. coli with plasmid DNA – Clues from the influence of ethanol
