IGEM:Paris Bettencourt 2012/Protocols/Heat Shock Transformation of E Coli

From OpenWetWare

Jump to: navigation, search

This protocol can be used to transform chemically competent E.Coli (i. e. from CaCl2) with a miniprepped plasmid or a ligation product.

Note: Never vortex competent cells.
Mix cells with gentle shaking.
  1. Thaw competent cells on ice.
    • These can be prepared using the CaCl2 protocol.
  2. Place 20 ul of cells in a pre-chilled Eppendorf tube.
  3. The next step depends on what is being transformed:
    • For an Intact Vector: Add 0.5 ul or less to the chilled cells
    • For a Ligation Product: Add 2-3 ul to the chilled cells
  4. Mix gently by flicking the tube.
  5. Chill on ice for 10 minutes.
    • This step is optional, but can improve yields when transforming a ligation product.
  6. Heat shock at 42°С for 30 seconds.
  7. Return to ice for 30 minutes.
  8. Add 200 ul LB medium and recover the cells by shaking at 37°С.
    • Another rich medium can substitute for the recovery.
    • The recovery time varies with the antibiotic selection.
      Ampicilin: 15-30 minutes.
      Kanamycin or Spectinomycin: 30-60 minutes
      Chloramphenicol: 60-120 minutes
  9. Plate out the cells on selective LB.
    • Use glass beads to spread the cells.
    • The volume of cells plated depends on what is being transformed:
      For an Intact Vector: High transformation efficiencies are expected. Plating out 10 ul of recovered cells should produce many colonies.
      For a Ligation Product: Lower transformation efficiencies are expected. Therefore you can plate the entire 200 ul volume of recovered cells.
    • Note: 200 ul is the maximum volume of liquid that an LB plate can absorb.
  10. Incubate at 37°С. Transformants should appear within 12 hrs.
Personal tools