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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=====
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	+	*[[Melbourne/1.7ml microcentrifuge|microcentrifuge: 1.7ml]]
	*Ice box		*Ice box
	*Pipettes		*Pipettes
Line 36:		Line 37:
	*Bunsen burner		*Bunsen burner
	*Spreader		*Spreader
		+	*[[Melbourne/primary ice|ice]]
		+
	=====References=====		=====References=====
	*		*

---

Latest revision as of 11:06, 29 September 2007

<Return to list of protocols> <Team home page>

• 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

• microcentrifuge: 1.7ml
• Ice box
• Pipettes
• 42 degree water bath (balance room)
• 37 degree incubator
• Bunsen burner
• Spreader
• ice

References

<Back to Protocols page>