Phenol/chloroform extraction - Revision history

Jakob Suckale: illustration

2010-09-13T13:37:15Z

illustration

←Older revision		Revision as of 13:37, 13 September 2010
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	==Notes==		==Notes==
		+	[[Image:Phenol chloroform gDNA ex tail vs liver.JPG\|thumb\|right\|200px\|Phenol/chloroform extraction of mostly genomic DNA from different tissue samples. Left: mouse tail sample; right: parallel extraction from mouse liver sample.]]
		+
	*Equilibrated phenol can typically be purchased from commercial sources. Alternatively, you can equilibrate it yourself. Be advised that this is NOT a fun procedure to carry out. The pH is important since chromosomal DNA will end up in the phenol phase if the pH is acid (arround pH 5). This fact can be used for [[RNA extraction]] but it is not a good idea to use acid phenol to clean your chromosomal DNA from proteins.		*Equilibrated phenol can typically be purchased from commercial sources. Alternatively, you can equilibrate it yourself. Be advised that this is NOT a fun procedure to carry out. The pH is important since chromosomal DNA will end up in the phenol phase if the pH is acid (arround pH 5). This fact can be used for [[RNA extraction]] but it is not a good idea to use acid phenol to clean your chromosomal DNA from proteins.
	*Phenol can undergo oxidation. Oxidized phenol will appear yellow or red in color (instead of clear) when saturated with water or buffer.		*Phenol can undergo oxidation. Oxidized phenol will appear yellow or red in color (instead of clear) when saturated with water or buffer.

Aurimas Vinckevicius: /* Notes */

2010-05-13T18:37:26Z

Notes

←Older revision		Revision as of 18:37, 13 May 2010
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	==Notes==		==Notes==
-	*Equilibrated phenol can typically be purchased from commercial sources. Alternatively, you can equilibrate it yourself. Be advised that this is NOT a fun procedure to carry out. The pH is important since chromosomal DNA will end up in the phenol phase if the pH is acid (arround pH 5). This fact can be used for [[RNA extraction]] but it is not a good idea to use acid phenol to clean your chromosomal DNA from proteins.	+	*Equilibrated phenol can typically be purchased from commercial sources. Alternatively, you can equilibrate it yourself. Be advised that this is NOT a fun procedure to carry out. The pH is important since chromosomal DNA will end up in the phenol phase if the pH is acid (arround pH 5). This fact can be used for [[RNA extraction]] but it is not a good idea to use acid phenol to clean your chromosomal DNA from proteins.
		+	*Phenol can undergo oxidation. Oxidized phenol will appear yellow or red in color (instead of clear) when saturated with water or buffer.
	*Phenol and chloroform should be used in a hood if possible.		*Phenol and chloroform should be used in a hood if possible.
	*Phenol is a dangerous substance that will burn you if it gets on your skin. WEAR GLOVES and BE CAREFUL. Check out the MSDS to verify the precautions you should take. A solution of PEG 400 is recommended for first aid. Phenol is both a systemic and local toxic agent. You want to visit the medical center.		*Phenol is a dangerous substance that will burn you if it gets on your skin. WEAR GLOVES and BE CAREFUL. Check out the MSDS to verify the precautions you should take. A solution of PEG 400 is recommended for first aid. Phenol is both a systemic and local toxic agent. You want to visit the medical center.

Jakob Suckale: illustration

2009-06-17T17:35:42Z

illustration

←Older revision		Revision as of 17:35, 17 June 2009
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	{{back to protocols}}		{{back to protocols}}
		+
	== General Information ==		== General Information ==
	[[Phenol]]/chloroform extraction is an easy way to remove proteins from your nucleic acid samples and can be carried out in a manner that is very close to quantitative. Nucleic acids remain in the aqueous phase and proteins separate into the organic phase or lie at the phase interface.		[[Phenol]]/chloroform extraction is an easy way to remove proteins from your nucleic acid samples and can be carried out in a manner that is very close to quantitative. Nucleic acids remain in the aqueous phase and proteins separate into the organic phase or lie at the phase interface.

	== General Procedure ==		== General Procedure ==
		+	[[Image:DNA extraction w phenol chloroform.jpg\|thumb\|right\|200px\|DNA extraction with phenol/chloroform/isoamyl alcohol pH 8 - aqueous top phase contains the majority of DNA, interphase mostly proteins, and lower organic phase most of the RNA and lipids]]
		+
	It is typically easiest to carry the extraction out in 1.7–2 mL eppendorf tubes.		It is typically easiest to carry the extraction out in 1.7–2 mL eppendorf tubes.

Torsten Waldminghaus at 17:28, 23 February 2009

2009-02-23T17:28:48Z

←Older revision		Revision as of 17:28, 23 February 2009
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		+	{{back to protocols}}
	== General Information ==		== General Information ==
	[[Phenol]]/chloroform extraction is an easy way to remove proteins from your nucleic acid samples and can be carried out in a manner that is very close to quantitative. Nucleic acids remain in the aqueous phase and proteins separate into the organic phase or lie at the phase interface.		[[Phenol]]/chloroform extraction is an easy way to remove proteins from your nucleic acid samples and can be carried out in a manner that is very close to quantitative. Nucleic acids remain in the aqueous phase and proteins separate into the organic phase or lie at the phase interface.

Torsten Waldminghaus: added some notes

2008-03-04T11:22:22Z

added some notes

Reshma P. Shetty at 01:41, 9 May 2007

2007-05-09T01:41:57Z

←Older revision		Revision as of 01:41, 9 May 2007
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	[[Protocols\|Return to Protocols]]		[[Protocols\|Return to Protocols]]

-	[[Category:Protocol]]	+	[[Category:Protocol]] [[Category:DNA]] [[Category:RNA]] [[Category:In vitro]]

Skosuri at 01:12, 29 November 2006

2006-11-29T01:12:16Z

←Older revision		Revision as of 01:12, 29 November 2006
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	== General Information ==		== General Information ==
-	Phenol/chloroform extraction is an easy way to remove proteins from your nucleic acid samples and can be carried out in a manner that is very close to quantitative. Nucleic acids remain in the aqueous phase and proteins separate into the organic phase or lie at the phase interface.	+	[[Phenol]]/chloroform extraction is an easy way to remove proteins from your nucleic acid samples and can be carried out in a manner that is very close to quantitative. Nucleic acids remain in the aqueous phase and proteins separate into the organic phase or lie at the phase interface.

	== General Procedure ==		== General Procedure ==
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	==Reagents==		==Reagents==
-	*Phenol equilibrated to pH 7.5	+	*[[Phenol]] equilibrated to pH 7.5
	*Chloroform:isoamyl alcohol in a 24:1 ratio (CHIZAM! in [http://www.scripps.edu/mb/joyce/ Joyce lab] lingo)		*Chloroform:isoamyl alcohol in a 24:1 ratio (CHIZAM! in [http://www.scripps.edu/mb/joyce/ Joyce lab] lingo)

Jenny T Nguyen at 16:25, 10 July 2006

2006-07-10T16:25:56Z

←Older revision		Revision as of 16:25, 10 July 2006
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	[[Protocols\|Return to Protocols]]		[[Protocols\|Return to Protocols]]
		+
		+	[[Category:Protocol]]

Tk: /* Notes */

2005-11-17T01:32:14Z

Notes

←Older revision		Revision as of 01:32, 17 November 2005
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	*Equilibrated phenol can typically be purchased from commercial sources. Alternatively, you can equilibrate it yourself. Be advised that this is NOT a fun procedure to carry out.		*Equilibrated phenol can typically be purchased from commercial sources. Alternatively, you can equilibrate it yourself. Be advised that this is NOT a fun procedure to carry out.
	*Phenol and chloroform should be used in a hood if possible.		*Phenol and chloroform should be used in a hood if possible.
-	*Phenol is a dangerous substance that will burn you if it gets on your skin. WEAR GLOVES and BE CAREFUL. Check out the MSDS to verify the precautions you should take.	+	*Phenol is a dangerous substance that will burn you if it gets on your skin. WEAR GLOVES and BE CAREFUL. Check out the MSDS to verify the precautions you should take. A solution of PEG 400 is recommended for first aid. Phenol is both a systemic and local toxic agent. You want to visit the medical center.
	*If you're in a hurry, you can shorten the protocol to two phenol extractions and one chloroform extraction.		*If you're in a hurry, you can shorten the protocol to two phenol extractions and one chloroform extraction.
-	*There are also commercial sources of phenol and chloroform mixed together and equilibrated. These are also sufficient for extraction, and I would recommend doing at least two extractions if you decide to go this route.	+	*There are also commercial sources of phenol and chloroform mixed together and equilibrated. These are also sufficient for extraction, and I would recommend doing at least two extractions if you decide to go this route. Phenol:Chloroform:Isoamyl alcohol usually has an upper layer of buffer saturated water in the bottle. Do not use this buffer layer -- you want the phenol/chloroform layer underneath.
		+	*Be careful to determine which layer is the phenol. The density of pure phenol (unlike phenol:chloroform:isoamyl alcohol) is almost 1.0. Small changes in the density of your water layer (excess salt, e.g.) can lead to layer inversion.
		+	*Removing the lower layer first can make it easier to recover the upper layer
		+	*Recovery can be improved by "washing" the upper layer by adding water, vortexing, recentrifuging, and recovering the water layer again.

	[[Protocols\|Return to Protocols]]		[[Protocols\|Return to Protocols]]

Kathmc at 03:07, 16 November 2005

2005-11-16T03:07:35Z

←Older revision		Revision as of 03:07, 16 November 2005
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	*If you're in a hurry, you can shorten the protocol to two phenol extractions and one chloroform extraction.		*If you're in a hurry, you can shorten the protocol to two phenol extractions and one chloroform extraction.
	*There are also commercial sources of phenol and chloroform mixed together and equilibrated. These are also sufficient for extraction, and I would recommend doing at least two extractions if you decide to go this route.		*There are also commercial sources of phenol and chloroform mixed together and equilibrated. These are also sufficient for extraction, and I would recommend doing at least two extractions if you decide to go this route.
		+
		+	[[Protocols\|Return to Protocols]]

←Older revision		Revision as of 11:22, 4 March 2008
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	==Reagents==		==Reagents==
-	*[[Phenol]] equilibrated to pH 7.5	+	*[[Phenol]] equilibrated to pH 7.5 (pH is important, see Notes below)
	*Chloroform:isoamyl alcohol in a 24:1 ratio (CHIZAM! in [http://www.scripps.edu/mb/joyce/ Joyce lab] lingo)		*Chloroform:isoamyl alcohol in a 24:1 ratio (CHIZAM! in [http://www.scripps.edu/mb/joyce/ Joyce lab] lingo)

	==Notes==		==Notes==
-	*Equilibrated phenol can typically be purchased from commercial sources. Alternatively, you can equilibrate it yourself. Be advised that this is NOT a fun procedure to carry out.	+	*Equilibrated phenol can typically be purchased from commercial sources. Alternatively, you can equilibrate it yourself. Be advised that this is NOT a fun procedure to carry out. The pH is important since chromosomal DNA will end up in the phenol phase if the pH is acid (arround pH 5). This fact can be used for [[RNA extraction]] but it is not a good idea to use acid phenol to clean your chromosomal DNA from proteins.
	*Phenol and chloroform should be used in a hood if possible.		*Phenol and chloroform should be used in a hood if possible.
	*Phenol is a dangerous substance that will burn you if it gets on your skin. WEAR GLOVES and BE CAREFUL. Check out the MSDS to verify the precautions you should take. A solution of PEG 400 is recommended for first aid. Phenol is both a systemic and local toxic agent. You want to visit the medical center.		*Phenol is a dangerous substance that will burn you if it gets on your skin. WEAR GLOVES and BE CAREFUL. Check out the MSDS to verify the precautions you should take. A solution of PEG 400 is recommended for first aid. Phenol is both a systemic and local toxic agent. You want to visit the medical center.
-	*If you're in a hurry, you can shorten the protocol to two phenol extractions and one chloroform extraction.	+	*If you're in a hurry, you can shorten the protocol to two phenol extractions and one chloroform extraction. The number of repetitions also depends on what kind of sample you have and what you want to do with it. If you have whole cell extracts you want to use more phenol steps as if you have only one restriction enzyme to get rid of. In the same way you might not care about residual phenol if you just want so run your DNA or RNA on a gel so one chlorophorm step is sufficient. Some other reactions you can do with nucleic acids are more sensitive to phenol so you should use chlorophorm two times.
	*There are also commercial sources of phenol and chloroform mixed together and equilibrated. These are also sufficient for extraction, and I would recommend doing at least two extractions if you decide to go this route. Phenol:Chloroform:Isoamyl alcohol usually has an upper layer of buffer saturated water in the bottle. Do not use this buffer layer -- you want the phenol/chloroform layer underneath.		*There are also commercial sources of phenol and chloroform mixed together and equilibrated. These are also sufficient for extraction, and I would recommend doing at least two extractions if you decide to go this route. Phenol:Chloroform:Isoamyl alcohol usually has an upper layer of buffer saturated water in the bottle. Do not use this buffer layer -- you want the phenol/chloroform layer underneath.
	*Be careful to determine which layer is the phenol. The density of pure phenol (unlike phenol:chloroform:isoamyl alcohol) is almost 1.0. Small changes in the density of your water layer (excess salt, e.g.) can lead to layer inversion.		*Be careful to determine which layer is the phenol. The density of pure phenol (unlike phenol:chloroform:isoamyl alcohol) is almost 1.0. Small changes in the density of your water layer (excess salt, e.g.) can lead to layer inversion.
	*Removing the lower layer first can make it easier to recover the upper layer		*Removing the lower layer first can make it easier to recover the upper layer
	*Recovery can be improved by "washing" the upper layer by adding water, vortexing, recentrifuging, and recovering the water layer again.		*Recovery can be improved by "washing" the upper layer by adding water, vortexing, recentrifuging, and recovering the water layer again.
		+	*Some people use pure chlorophorm instead of chloroform:isoamyl alcohol

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	[[Category:Protocol]] [[Category:DNA]] [[Category:RNA]] [[Category:In vitro]]		[[Category:Protocol]] [[Category:DNA]] [[Category:RNA]] [[Category:In vitro]]