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

This protocol can be used to transform chemically competent E.Coli (i. e. from [math]\displaystyle{ CaCl_{2} }[/math]) 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 [math]\displaystyle{ CaCl_{2} }[/math] 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.