IGEM:IMPERIAL/2006/Protocols/transformation

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Transformation

  • Carry out the modified glass milk purification.
  • Take out E.coli DH5-α from -80[[:Category:{{{1}}}|{{{1}}}]] and thaw on ice.
  • Put cuvettes on ice.
  • Switch on the electroporation machine. Set to 25, 200 (black knob) and set the voltage to 2.2-2.3. Calibrate by empty zapping.
  • Take 1μL of DNA and add it into the 50μL aliquot of E.coli. Set the Gilson to about 60μL and mix carefully and transfer into chilled cuvettes. Make sure not to get bubbles! If you get this wrong or there is too much salt it will spark. If you have a spark then the cuvette must be thrown away.
  • Set up the cuvettes on the electroporation machine (make sure bottom is covered with mixture) and zap!
  • Remove the zapped cells from the cuvette and transfer back to the eppendorf.
  • Add 0.5 mL LB and plate the entire volume. It is okay to throw the eppendorf away at this point.
  • Plate it onto LB amp and leave in 37[[:Category:{{{1}}}|{{{1}}}]] O/N.
  • Clean cuvettes thoroughly (10x under warm water, followed by 3x in distilled water) and dry in the 37[[:Category:{{{1}}}|{{{1}}}]] incubator with plates
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