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 CaCl2) with a miniprepped plasmid or a ligation product.
- Thaw competent cells on ice.
- These can be prepared using the CaCl2 protocol.
- Place 20 ul of cells in a pre-chilled Eppendorf tube.
- 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
- Mix gently by flicking the tube.
- Chill on ice for 10 minutes.
- This step is optional, but can improve yields when transforming a ligation product.
- Heat shock at 42°С for 30 seconds.
- Return to ice for 30 minutes.
- 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
- 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.
- Incubate at 37°С. Transformants should appear within 12 hrs.