Team:BIOTEC Dresden/ProtocolsAHL assay


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  • Escherichia coli DH5 alpha chemical competent cells
  • 1mL SOC (room temperature) for each reaction
  • Ice
  • Plasmid DNA
  • Heating block
  • 1.5ml microfuge tube
  • LB-agar plate with corresponding antibiotic


  • Chill DNA samples and tubes on ice.
  • Place LB-agar plates in 37°C incubator to warm.
  • Remove chemical competent cells from -80°C freezer and thaw on ice. Alternatively, freshly prepared chemical competent cells may be used immediately.
  • Dial a P2 pipetman to either 1 or 2μL depending on the salt content of your DNA sample. Use 2μL for samples that have been purified in some way.
  • Dial a P200 pipetman to 50μL or whatever volume of chemical competent cells you want to use; usually 20-50μL .
  • Pipet 1-2μL of DNA sample and add to chemical competent cells. Swirl tip around gently in cells to mix DNA and cells. Do not pipet up and down.
  • Place the mix back on ice for 30 mins.
  • Pulse the cells with a heat shock by placing the microfuge tubes in a heating block for 45 seconds at 42°C.
  • Place sample on ice for 2 mins and then add SOC medium. This step should be done as quickly as possible to prevent cells from dying off.
  • Chill sample on ice for 2 mins to permit the cells to recover.
  • Transfer eppendorf tube to 37°C incubator and shake to promote aeration. Incubate for 1 hr to permit expression of antibiotic resistance gene.
  • Plate transformation onto prewarmed LB-agar plate supplemented with appropriate antibiotic.


adapted from

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