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Team:Cambridge/Protocols/Transformation of E.Coli
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| + | The key precautions are to wear gloves and a lab coat, and aim to perform good sterile technique this will keep your plates uncontaminated and reduce the chance of you yourself coming into contact with the bacteria or any chemicals on the bench top. | ||
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Revision as of 17:28, 12 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
How it works
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 (ul) |
| Competent E.coli cells | 50 |
| DNA | 10 |
Incubate the cells with the DNA at 42 degrees C for 1 minute.
Add 250ul of liquid broth (which should have already been autoclaved)
Incubate at 37 degrees C for 1 hour.
Plate 10ul on agar containing an antibiotic (the antibiotic for which resistance is included in the DNA being taken up by the cells) and also plate 100ul 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 37 degrees C
Always keep agar plates upside down so that drips of condensation and falling debris does not contaminate them.
Safety
The E.coli strains we use are non-pathogenic, so the material itself is not hazardous to humans. The key precautions are to wear gloves and a lab coat, and aim to perform good sterile technique this will keep your plates uncontaminated and reduce the chance of you yourself coming into contact with the bacteria or any chemicals on the bench top.
