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Transformation of Bacteria

Revision as of 15:44, 12 February 2014 by Davebridges (Talk | contribs) (updated transformation protocol)

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Revision as of 15:44, 12 February 2014 by Davebridges (Talk | contribs) (updated transformation protocol)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

Materials

  • Competent Cells
  • SOC Buffer or LB Media
  • DNA of interest (1 uL for plasmid, 5 uL for cloning/mutagenesis)
  • Plates (Amp or Kan; in cold room, see Making LB Agar Plates )

Protocol

  1. Thaw cells on ice and label
  2. Add DNA to cells and mix by tapping
  3. Incubate on ice 30-45min
  4. Heat shock at 42C for 45s
  5. Place back on ice
  6. Add 450 uL of SOC Buffer or LB.
  7. Incubate at 37C for 1h with occasional mixing
  8. Plate 30-50 uL for amplification, or all for cloning/mutagenesis
  • When plating be sure to label plates and use extreme caution when sterilizing the spreader with EtOH and fire.
Last modified on 12 February 2014, at 15:44