Team:Cambridge/Protocols/Transformation of E.Coli

Transformation of E.coli

A simple method of transforming competent E.coli cells with your DNA of choice.

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 0^oC with CaCl₂ solution, adding the DNA and heat shocking the bacteria for a short period of time, allowing the cells to recover at 37^oC and then plate them.

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.

Preparation

Ensure that you have enough sterilized liquid broth (or another medium such as SOC) and agar plates containing the correct antibiotic. Before you begin have a waterbath set to 42^oC and an incubator at 37^oC.

Practice

Take care not to disturb the competent E.coli: do not vortex them or pipette them up and down.

• Thaw competent E.coli cells ( 50 μl in an eppendorf tube, grown to an OD₆₀₀ of 0.2 - 0.5) on ice. Let them sit on ice for at least 10 minutes.
• Add 1 μl of plasmid and mix gently by stirring with the pipette tip. Use more plasmid solution if it is very weak. We have had success with about 50 ng of plasmid DNA.
• Incubate the cells with the DNA at 42ºC for 1 minute.
• Add 250 μl of sterile liquid broth.

At this stage it is important to maintain sterility to avoid contaminating the liquid broth or the plates. Do not allow the pipette tip to touch anything before pipetting the liquid broth. Leaving the liquid broth bottle on the bench overnight should reveal any contamination in the morning.

• Incubate at 37ºC for 1 hour.
• Plate 10μl on agar containing an antibiotic (the antibiotic for which the plasmid confers resistance) and also plate 100μl on another identical plate. This allows for possible overcrowding so that single colonies can 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.

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