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Transformation of Bacteria

Revision as of 15:05, 5 May 2009 by Jpecherer (Talk | contribs) (Update)

Revision as of 15:05, 5 May 2009 by Jpecherer (Talk | contribs) (Update)

Materials

  • Competent Cells
  • Plasmid amplification use subcloning efficiency DH5a
  • Cloning use OneShot TOP10 (Pink)
  • Mutagenesis use XL1 Blue Supercompetent (Blue), comes in 50uL aliquots
  • SOC Buffer
  • DNA of interest (1 uL for plasmid, 5 uL for cloning/mutagenesis)
  • Plates (Amp or Kan; in cold room)

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 on shelf OR add 1000uL and adjust the level.
  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