Melbourne/Transformation Protocol
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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===== | ||
| - | *Competent cells (from -70degree freezer) | + | *[[Melbourne DH5a|Competent cells]] (from -70degree freezer) |
*DNA for transformation | *DNA for transformation | ||
| - | *LB (cupboard) | + | *[[Melbourne/Secondary Reagent LB|LB]] (cupboard) |
| - | *LB-agar plates with selective antibiotic (cool room) | + | *[[Melbourne/Secondary Reagent Agar Plates|LB-agar plates]] with selective antibiotic (cool room) |
=====Method including controls===== | =====Method including controls===== | ||
Latest revision as of 11:06, 29 September 2007
<Return to list of protocols> <Team home page>
- Applications:
- Amplification of Biobrick DNA for storage and use.
- 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
- Competent cells (from -70degree freezer)
- DNA for transformation
- LB (cupboard)
- LB-agar plates with selective antibiotic (cool room)
Method including controls
- Add 1uL resuspended plasmid DNA to 50uL competent cells.
- Incubate on ice for 45min.
- Heat shock in water bath at 42 degrees for 1min.
- Incubate on ice for 15min.
- Add 1mL LB (flame tip before use).
- Incubate at 37 degrees for 1 hour.
- Spin down cells and remove majority of LB.
- Resuspend cells in remaining LB.
- Under a bunsen spread resuspended bacteria on agar plate on selective antibiotic.
- Incubate plate overnight at 37 degrees.
- Place in cold room until needed.
Equipement Required
- microcentrifuge: 1.7ml
- Ice box
- Pipettes
- 42 degree water bath (balance room)
- 37 degree incubator
- Bunsen burner
- Spreader
- ice
References
