Melbourne/Transformation Protocol shorter

From 2007.igem.org

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(Primary & secondary Reagents Required including controls)
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====Method from primary and secondary reagents====
====Method from primary and secondary reagents====
=====Primary & secondary Reagents Required including controls=====
=====Primary & secondary Reagents Required including controls=====
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*Competent cells (from -70degree freezer)
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*[[Melbourne DH5a|Competent cells (from -70degree freezer)]]
*DNA for transformation
*DNA for transformation
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*LB (cupboard)
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*[[Melbourne/Secondary Reagent LB|LB]] (cupboard)
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*LB-agar plates with selective antibiotic (cool room)
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*[[Melbourne/Secondary Reagent Agar Plates|LB-agar plates]] with selective antibiotic (cool room)
=====Method including controls=====
=====Method including controls=====

Revision as of 11:44, 28 September 2007

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  • Applications:
    1. Amplification of Biobrick DNA for storage and use.
    2. Selection and 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
Method including controls
  1. Add 1uL resuspended plasmid DNA to 50uL competent cells.
  2. Incubate on ice for 30min.
  3. Heat shock in water bath at 42 degrees for 1min.
  4. Incubate on ice for 10min.
  5. Add 1mL LB (flame tip before use).
  6. Incubate at 37 degrees for 30 minutes.
  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 (balance room)
  • 37 degree incubator
  • Bunsen burner
  • Spreader
References


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