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-	Inoculate 5mL of LB medium with appropriate antibody (e.g. 5 microliter of ampicillin) with a single bacterial colony and incubate at 37 degrees C overnight with vigorous shaking.	+	[[Melb:Protocols for Standard Methods |<Return to list of protocols>]]  [[Melbourne|  <Team home page>]]
		+	*Applications:
		+	*#Purification of plasmid DNA from bacterial cultures
-	Pour 1.5mL of the above culture into an Eppendorf tube. Centrifuge for 1 min. at 10,000g and discard the supernatant by aspiration.	+	*Time to complete protocol:
		+	**Lab time: 3h
		+	**Waiting time: 30min, 15min, 1hour, overnight.
		+	*Approximate cost of materials: $
		+	====Method from primary and secondary reagents====
		+	=====Primary & secondary Reagents Required including controls=====
		+	*Bacterial colony
		+	*appropriate antibiotic
		+	*[[Melbourne/Secondary Reagent LB|LB]] (cupboard)
		+	*[[Melbourne/primary EDTA|EDTA]]
		+	*[[Melbourne/primary glycerol|Glycerol]]
		+	*[[Melbourne/primary Tris|TRIS]]
		+	*NaOH
		+	*SDS
		+	*Potassium Acetate
		+	*Phenol
		+	*Chloroform
		+	*100% Ethanol
		+	*70% Ethanol
		+	*[[Melbourne/primary milliq|milliQ water]]
		+	*RNAse
-	Resuspend the pellet by vortexing  in 100 microliters of ice- cold Solution A:	+	=====Method including controls=====
		+
		+
		+	#Inoculate 5mL of LB medium with appropriate antibiotic (e.g. 5 microliter of ampicillin) with a single bacterial colony and incubate at 37 degrees C overnight with vigorous shaking.
		+
		+	#Pour 1.5mL of the above culture into an Eppendorf tube. Centrifuge for 1 min. at 10,000g and discard the supernatant by aspiration/decanting.
		+
		+
		+	#Resuspend the pellet by vortexing  in 100ul of ice-cold Solution A:
	50mM Glucose		50mM Glucose
	10mM EDTA		10mM EDTA
	25mM Tris Cl (pH 8.0)		25mM Tris Cl (pH 8.0)
-	Add 200 microliters of freshly prepared Solution B:	+	#Add 200 microliters of freshly prepared Solution B:
	0.2M NaOH		0.2M NaOH
	1% SDS		1% SDS
Line 15:		Line 45:
	invert rapidly 4 times. No vortexing.		invert rapidly 4 times. No vortexing.
-	Add 150 microliters of ice-cold Solution C:	+	#Add 150 microliters of ice-cold Solution C:
	3M potassium		3M potassium
	5M acetate		5M acetate
	(pH 4.8)		(pH 4.8)
-	vortex for 10 seconds and place on ice for 15 minutes.	+	#vortex for 10 seconds and place on ice for 15 minutes.
-	Transfer the supernatant to a fresh tube.	+	#Transfer the supernatant to a fresh tube.
-	Add an equal amount (400 microliters) of phenol/chroloform (200/200 microliters).	+	#Add an equal amount (400ul) of phenol/chroloform (200/200ul).
	Vortex briefly and centrifuge at 10,000g for 2 minutes.		Vortex briefly and centrifuge at 10,000g for 2 minutes.
-	Transfer the supernatant to a fresh tube.	+	#Transfer the supernatant to a fresh tube.
-	Add two volumes (800 microliters) of 100% ethanol. Vortex and place on ice for 20 minutes.	+	#Add two volumes (800ul) of 100% ethanol. Vortex and place on ice for 20 minutes.
	Centrifuge at 10,000g for 15 minutes. Discard the supernatant.		Centrifuge at 10,000g for 15 minutes. Discard the supernatant.
-	Wash by adding 400 microliters of 70% ethanol. Centrifuge for 10 minutes.	+	#Wash by adding 400ul of 70% ethanol. Centrifuge for 10 minutes.
	Discard the supernatant and allow the tube to dry (e.g. vacuum dessicator).		Discard the supernatant and allow the tube to dry (e.g. vacuum dessicator).
-	Add 30 microliters of TE buffer with a small amount of RNAase.	+	#Add 30ul of TE buffer (or water) with a small amount of RNAase.
		+
		+	=====Equipement Required=====
		+	*[[Melbourne/1.7ml microcentrifuge|microcentrifuge: 1.7ml]]
		+	*Ice box
		+	*Pipettes
		+	*[[Melbourne/primary ice|ice]]

---

Revision as of 12:53, 28 September 2007

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

• Applications:
  1. Purification of plasmid DNA from bacterial cultures

• Time to complete protocol:
  • Lab time: 3h
  • Waiting time: 30min, 15min, 1hour, overnight.
• Approximate cost of materials: $

Contents 1 Method from primary and secondary reagents 1.1 Primary & secondary Reagents Required including controls 1.2 Method including controls 1.3 Equipement Required

Method from primary and secondary reagents

Primary & secondary Reagents Required including controls

• Bacterial colony
• appropriate antibiotic
• LB (cupboard)
• EDTA
• Glycerol
• TRIS
• NaOH
• SDS
• Potassium Acetate
• Phenol
• Chloroform
• 100% Ethanol
• 70% Ethanol
• milliQ water
• RNAse

Method including controls

1. Inoculate 5mL of LB medium with appropriate antibiotic (e.g. 5 microliter of ampicillin) with a single bacterial colony and incubate at 37 degrees C overnight with vigorous shaking.

1. Pour 1.5mL of the above culture into an Eppendorf tube. Centrifuge for 1 min. at 10,000g and discard the supernatant by aspiration/decanting.

1. Resuspend the pellet by vortexing in 100ul of ice-cold Solution A:

50mM Glucose 10mM EDTA 25mM Tris Cl (pH 8.0)

1. Add 200 microliters of freshly prepared Solution B:

0.2M NaOH 1% SDS

invert rapidly 4 times. No vortexing.

1. Add 150 microliters of ice-cold Solution C:

3M potassium 5M acetate (pH 4.8)

1. vortex for 10 seconds and place on ice for 15 minutes.

1. Transfer the supernatant to a fresh tube.

1. Add an equal amount (400ul) of phenol/chroloform (200/200ul).

Vortex briefly and centrifuge at 10,000g for 2 minutes.

1. Transfer the supernatant to a fresh tube.

1. Add two volumes (800ul) of 100% ethanol. Vortex and place on ice for 20 minutes.

Centrifuge at 10,000g for 15 minutes. Discard the supernatant.

1. Wash by adding 400ul of 70% ethanol. Centrifuge for 10 minutes.

Discard the supernatant and allow the tube to dry (e.g. vacuum dessicator).

1. Add 30ul of TE buffer (or water) with a small amount of RNAase.

Equipement Required

• microcentrifuge: 1.7ml
• Ice box
• Pipettes
• ice