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	*Applications:		*Applications:
	*#Amplification of Biobrick DNA for storage and use.		*#Amplification of Biobrick DNA for storage and use.
-	*#Selection aand amplification of ligated constructs	+	*#Selection and amplification of ligated constructs.
	*Time to complete protocol:		*Time to complete 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	+	*Competent cells (from -70degree freezer)
	*DNA for transformation		*DNA for transformation
-	*LB	+	*LB (cupboard)
-	*LB-agar plates with selective antibiotic	+	*LB-agar plates with selective antibiotic (cool room)
	=====Method including controls=====		=====Method including controls=====
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	*Ice box		*Ice box
	*Pipettes		*Pipettes
-	*42 degree water bath	+	*42 degree water bath (balance room)
-	*37 degree incubator	+	*37 degree incubator
	*Bunsen burner		*Bunsen burner
	*Spreader		*Spreader

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Revision as of 05:30, 2 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.
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 (balance room)
• 37 degree incubator
• Bunsen burner
• Spreader

References

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