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Revision as of 22:07, 3 July 2007 (view source) PhillipDodson (Talk | contribs) m (Melb:Transformation Protocol moved to Melbourne/Transformation Protocol) ← Older edit		Revision as of 06:22, 4 July 2007 (view source) Patricia (Talk | contribs) (→Method including controls) Newer edit →
Line 21:		Line 21:
	#Heat shock in water bath at 42 degrees for 1min.		#Heat shock in water bath at 42 degrees for 1min.
	#Incubate on ice for 15min.		#Incubate on ice for 15min.
-	#Add 1mL LB.	+	#Add 1mL LB (flame tip before use).
-	#Incubate at 37degrees for 1 hour.	+	#Incubate at 37 degrees for 1 hour.
	#Spin down cells and remove majority of LB.		#Spin down cells and remove majority of LB.
	#Resuspend cells in remaining LB.		#Resuspend cells in remaining LB.
Line 28:		Line 28:
	#Incubate plate overnight at 37 degrees.		#Incubate plate overnight at 37 degrees.
	#Place in cold room until needed.		#Place in cold room until needed.
		+
	=====Equipement Required=====		=====Equipement Required=====
	*1.5mL Microfuge tubes		*1.5mL Microfuge tubes

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Revision as of 06:22, 4 July 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

• Competent cells (from -70degree freezer)
• DNA for transformation
• LB (cupboard)
• LB-agar plates with selective antibiotic (cool room)

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 (flame tip before use).
6. Incubate at 37 degrees 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 (balance room)
• 37 degree incubator
• Bunsen burner
• Spreader

References

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