Melbourne/Transformation Protocol

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Revision as of 05:27, 2 July 2007

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• Applications:
  1. Amplification of Biobrick DNA for storage and use.
  2. Selection aand amplification of ligated constructs

• Time to complete protocol:
  • Lab time: 10min, 10min, 10min, 15min.
  • Waiting time: 45min, 15min, 1hour, overnight.
• Approximate cost of materials: $

Method from primary and secondary reagents

Primary & secondary Reagents Required including controls

• Competent cells
• DNA for transformation
• LB
• LB-agar plates with selective antibiotic

Method including controls

1. Add 1uL resuspended plasmid DNA to 50uL competent cells.
2. Incubate on ice for 45min.
3. Heat shock in water bath at 42 degrees for 1min.
4. Incubate on ice for 15min.
5. Add 1mL LB.
6. Incubate at 37degrees for 1 hour.
7. Spin down cells and remove majority of LB.
8. Resuspend cells in remaining LB.
9. Under a bunsen spread resuspended bacteria on agar plate on selective antibiotic.
10. Incubate plate overnight at 37 degrees.
11. Place in cold room until needed.

Equipement Required

• 1.5mL Microfuge tubes
• Ice box
• Pipettes
• 42 degree water bath
• 37 degree incubator
• Bunsen burner
• Spreader

References

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