Team:Newcastle/Transformation of E. coli

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Transformation of E. coli

Materials required

  • E. coli DH5α (200 µl)
  • Appropriate vector DNA
  • Heat block
  • Bucket of ice
  • Pipettes
  • Eppendorf tubes
  • 1.5% agar plate containing appropriate antibiotics

Protocol

  1. Thaw a 200 µl aliquot of E. coli DH4α and add the transforming DNA (10 ng of vector DNA in 10 µl).
  2. Incubate on ice for 30 minutes.
  3. Heat-shock the cells for 120 seconds at 42°C, and place on ice again for 3-4 minutes.
  4. Add 1 ml of LB broth and incubate the cells at 37°C for 1-1.5 hr in a water bath with gentle shaking.
  5. Plate 200 µl of transformed E. coli onto 1.5% agar plate containing the appropriate selection markers.
  6. Incubate the plates overnight at 37°C.


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