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Revision as of 12:59, 28 September 2007 (view source) Craig (Talk | contribs) (→Method including controls) ← Older edit		Latest revision as of 11:11, 29 September 2007 (view source) Craig (Talk | contribs) (→Primary & secondary Reagents Required including controls)
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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=====
-	*Bacterial colony	+	*Bacterial colony overnight culture
	*appropriate antibiotic		*appropriate antibiotic
	*[[Melbourne/Secondary Reagent LB|LB]] (cupboard)		*[[Melbourne/Secondary Reagent LB|LB]] (cupboard)
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	*[[Melbourne/primary glycerol|Glycerol]]		*[[Melbourne/primary glycerol|Glycerol]]
	*[[Melbourne/primary Tris|TRIS]]		*[[Melbourne/primary Tris|TRIS]]
-	*NaOH	+	*[[Melbourne/primary NaOH|NaOH]]
	*SDS		*SDS
	*Potassium Acetate		*Potassium Acetate
	*Phenol		*Phenol
	*Chloroform		*Chloroform
-	*100% Ethanol	+	*100% [[Melbourne/primary Ethanol|Ethanol]]
-	*70% Ethanol	+	*70% [[Melbourne/primary Ethanol|Ethanol]]
	*[[Melbourne/primary milliq|milliQ water]]		*[[Melbourne/primary milliq|milliQ water]]
	*RNAse		*RNAse

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Latest revision as of 11:11, 29 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 overnight culture
• 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.
2. 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.
3. Resuspend the pellet by vortexing in 100ul of ice-cold Solution A:
   -50mM Glucose
   -10mM EDTA
   -25mM Tris Cl (pH 8.0)
4. Add 200 microliters of freshly prepared Solution B:
   -0.2M NaOH
   -1% SDS
   invert rapidly 4 times. No vortexing.
5. Add 150 microliters of ice-cold Solution C:
   -3M potassium
   -5M acetate (pH 4.8)
6. vortex for 10 seconds and place on ice for 15 minutes.
7. Transfer the supernatant to a fresh tube.
8. Add an equal amount (400ul) of phenol/chroloform (200/200ul). Vortex briefly and centrifuge at 10,000g for 2 minutes.
9. Transfer the supernatant to a fresh tube.
10. 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.
11. Wash by adding 400ul of 70% ethanol. Centrifuge for 10 minutes. Discard the supernatant and allow the tube to dry (e.g. vacuum dessicator).
12. Add 30ul of TE buffer (or water) with a small amount of RNAase.

Equipement Required

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