Ethanol precipitation of nucleic acids - Revision history

Christopher C Vanlang: /* Source Code */

2012-07-01T10:48:58Z

Source Code

←Older revision		Revision as of 10:48, 1 July 2012
Line 68:		Line 68:
	[[Category:RNA]]		[[Category:RNA]]
	[[Category:In vitro]]		[[Category:In vitro]]
		+	[[Category:Purification]]

Christopher C Vanlang: /* 96 Well Plate Protocol */

2011-12-22T09:26:14Z

96 Well Plate Protocol

←Older revision		Revision as of 09:26, 22 December 2011
Line 29:		Line 29:
	#*Beware of using water unless you are sure of what you are getting in to. The "pH" of water can vary widely (I've seen from pH 5 to pH 8.5), and depurination of DNA at low pH or degradation of RNA at high pH are possibilities. Water also typically contains trace metals, which can accelerate these reactions. I typically recommend resuspension in TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). This makes sure your nucleic acid is at a neutral pH and the EDTA will chelate any trace metals. Since they are in such small amounts, neither the buffer nor the EDTA will affect most downstream reactions.--[[User:Kathmc\|Kathleen]]		#*Beware of using water unless you are sure of what you are getting in to. The "pH" of water can vary widely (I've seen from pH 5 to pH 8.5), and depurination of DNA at low pH or degradation of RNA at high pH are possibilities. Water also typically contains trace metals, which can accelerate these reactions. I typically recommend resuspension in TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). This makes sure your nucleic acid is at a neutral pH and the EDTA will chelate any trace metals. Since they are in such small amounts, neither the buffer nor the EDTA will affect most downstream reactions.--[[User:Kathmc\|Kathleen]]
	=== 96 Well Plate Protocol===		=== 96 Well Plate Protocol===
-	#*Add to each 10 µl product:	+	#Add to each 10 µl product:
-	**1.9 µl of Na acetate 3M	+	#*1.9 µl of Na acetate 3M
-	**60 µl of 85% ethanol	+	#*60 µl of 85% ethanol
-	#*Mix thoroughly (vortex ???) and keep at -20°C for 30 min	+	#Mix thoroughly (vortex ???) and keep at -20°C for 30 min
-	#*centrifuge for 45 min at 4000 rpm and 4°C (program 3; balance)	+	#centrifuge for 45 min at 4000 rpm and 4°C (program 3; balance)
-	#*remove supernatant (invert tube on trash once)	+	#remove supernatant (invert tube on trash once)
-	#*add 150 µl of 70% ethanol and mix	+	#add 150 µl of 70% ethanol and mix
-	#*centrifuge for 15 min at 4000 rpm and 4°C	+	#centrifuge for 15 min at 4000 rpm and 4°C
-	#*remove all supernatant (invert tube on trash)	+	#remove all supernatant (invert tube on trash)
-	#*invert tube on paper tissue	+	#invert tube on paper tissue
-	#*centrifuge for 2 min at 500 rpm	+	#centrifuge for 2 min at 500 rpm
-	#*take out the tube and let it dry in the fume hood at room temperature for 10-15 min	+	#take out the tube and let it dry in the fume hood at room temperature for 10-15 min
-	#*put 20 µl of formide dye using multipipette (nasty chemical to manipulate in fume hood)	+	#put 20 µl of formide dye using multipipette (nasty chemical to manipulate in fume hood)
-	#*vortex thoroughly/spin/vortex/spin	+	#vortex thoroughly/spin/vortex/spin
-	#*transfer the 20 µl (multipipette) in a sequencing plate	+	#transfer the 20 µl (multipipette) in a sequencing plate
-	#*put septum on top, press, tap once (do NOT mix)	+	#put septum on top, press, tap once (do NOT mix)
-	#*keep on ice in aluminium rack	+	#keep on ice in aluminium rack
-	#*Heat 3 min at 95°C (SWATI/95-CST) to keep in single stranded form)	+	#Heat 3 min at 95°C (SWATI/95-CST) to keep in single stranded form)
-	#*keep on ice in aluminium rack	+	#keep on ice in aluminium rack

	==Notes==		==Notes==

Christopher C Vanlang: /* 96 Well Plate Protocol */

2011-12-22T09:24:30Z

96 Well Plate Protocol

←Older revision		Revision as of 09:24, 22 December 2011
Line 29:		Line 29:
	#*Beware of using water unless you are sure of what you are getting in to. The "pH" of water can vary widely (I've seen from pH 5 to pH 8.5), and depurination of DNA at low pH or degradation of RNA at high pH are possibilities. Water also typically contains trace metals, which can accelerate these reactions. I typically recommend resuspension in TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). This makes sure your nucleic acid is at a neutral pH and the EDTA will chelate any trace metals. Since they are in such small amounts, neither the buffer nor the EDTA will affect most downstream reactions.--[[User:Kathmc\|Kathleen]]		#*Beware of using water unless you are sure of what you are getting in to. The "pH" of water can vary widely (I've seen from pH 5 to pH 8.5), and depurination of DNA at low pH or degradation of RNA at high pH are possibilities. Water also typically contains trace metals, which can accelerate these reactions. I typically recommend resuspension in TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). This makes sure your nucleic acid is at a neutral pH and the EDTA will chelate any trace metals. Since they are in such small amounts, neither the buffer nor the EDTA will affect most downstream reactions.--[[User:Kathmc\|Kathleen]]
	=== 96 Well Plate Protocol===		=== 96 Well Plate Protocol===
-	*Add to each 10 µl product:	+	#*Add to each 10 µl product:
-	- 1.9 µl of Na acetate 3M	+	**1.9 µl of Na acetate 3M
-	- 60 µl of 85% ethanol	+	**60 µl of 85% ethanol
-	*Mix thoroughly (vortex ???)	+	#*Mix thoroughly (vortex ???) and keep at -20°C for 30 min
-	*keep at -20°C for 30 min	+	#*centrifuge for 45 min at 4000 rpm and 4°C (program 3; balance)
-	*centrifuge for 45 min at 4000 rpm and 4°C (program 3; balance)	+	#*remove supernatant (invert tube on trash once)
-	*remove supernatant (invert tube on trash once)	+	#*add 150 µl of 70% ethanol and mix
-	*add 150 µl of 70% ethanol	+	#*centrifuge for 15 min at 4000 rpm and 4°C
-	*mix	+	#*remove all supernatant (invert tube on trash)
-	*centrifuge for 15 min at 4000 rpm and 4°C ~~(program 4; balance)~~	+	#*invert tube on paper tissue
-	*remove all supernatant (invert tube on trash)	+	#*centrifuge for 2 min at 500 rpm
-	*invert tube on paper tissue	+	#*take out the tube and let it dry in the fume hood at room temperature for 10-15 min
-	*centrifuge for 2 min at 500 rpm ~~(program 7; no need to balance)~~	+	#*put 20 µl of formide dye using multipipette (nasty chemical to manipulate in fume hood)
-	*take out the tube and let it dry in the fume hood at room temperature for 10-15 min	+	#*vortex thoroughly/spin/vortex/spin
-	*put 20 µl of formide dye using multipipette (nasty chemical to manipulate in fume hood)	+	#*transfer the 20 µl (multipipette) in a sequencing plate
-	*vortex thoroughly/spin/vortex/spin	+	#*put septum on top, press, tap once (do NOT mix)
-	*transfer the 20 µl (multipipette) in a ~~sequecing~~ plate ~~(careful on the order and remember that blocks of 4 are used)~~	+	#*keep on ice in aluminium rack
-	*put septum on top, press, tap once (do NOT mix)	+	#*Heat 3 min at 95°C (SWATI/95-CST) to keep in single stranded form)
-	*keep on ice in aluminium rack	+	#*keep on ice in aluminium rack
-	*Heat 3 min at 95°C (SWATI/95-CST) to keep in single stranded form)	+
-	*keep on ice in aluminium rack	+

	==Notes==		==Notes==

Christopher C Vanlang: /* Procedure */

2011-12-22T09:05:29Z

Procedure

←Older revision		Revision as of 09:05, 22 December 2011
Line 13:		Line 13:

	==Procedure==		==Procedure==
		+	===Eppendorf Protocol===
	[[Image:Tube placement in centrifuge.jpg\|thumb\|200px\|How to arrange tubes in the centrifuge.]]		[[Image:Tube placement in centrifuge.jpg\|thumb\|200px\|How to arrange tubes in the centrifuge.]]
	[[Image:DNA EtOH precipitation small pellet.jpg\|thumb\|200px\|Pellets can be very small and hard to see.]]		[[Image:DNA EtOH precipitation small pellet.jpg\|thumb\|200px\|Pellets can be very small and hard to see.]]
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	#Add your desired quantity of water. Vortex and spin down to resuspend.		#Add your desired quantity of water. Vortex and spin down to resuspend.
	#*Beware of using water unless you are sure of what you are getting in to. The "pH" of water can vary widely (I've seen from pH 5 to pH 8.5), and depurination of DNA at low pH or degradation of RNA at high pH are possibilities. Water also typically contains trace metals, which can accelerate these reactions. I typically recommend resuspension in TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). This makes sure your nucleic acid is at a neutral pH and the EDTA will chelate any trace metals. Since they are in such small amounts, neither the buffer nor the EDTA will affect most downstream reactions.--[[User:Kathmc\|Kathleen]]		#*Beware of using water unless you are sure of what you are getting in to. The "pH" of water can vary widely (I've seen from pH 5 to pH 8.5), and depurination of DNA at low pH or degradation of RNA at high pH are possibilities. Water also typically contains trace metals, which can accelerate these reactions. I typically recommend resuspension in TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). This makes sure your nucleic acid is at a neutral pH and the EDTA will chelate any trace metals. Since they are in such small amounts, neither the buffer nor the EDTA will affect most downstream reactions.--[[User:Kathmc\|Kathleen]]
		+	=== 96 Well Plate Protocol===
		+	*Add to each 10 µl product:
		+	- 1.9 µl of Na acetate 3M
		+	- 60 µl of 85% ethanol
		+	*Mix thoroughly (vortex ???)
		+	*keep at -20°C for 30 min
		+	*centrifuge for 45 min at 4000 rpm and 4°C (program 3; balance)
		+	*remove supernatant (invert tube on trash once)
		+	*add 150 µl of 70% ethanol
		+	*mix
		+	*centrifuge for 15 min at 4000 rpm and 4°C (program 4; balance)
		+	*remove all supernatant (invert tube on trash)
		+	*invert tube on paper tissue
		+	*centrifuge for 2 min at 500 rpm (program 7; no need to balance)
		+	*take out the tube and let it dry in the fume hood at room temperature for 10-15 min
		+	*put 20 µl of formide dye using multipipette (nasty chemical to manipulate in fume hood)
		+	*vortex thoroughly/spin/vortex/spin
		+	*transfer the 20 µl (multipipette) in a sequecing plate (careful on the order and remember that blocks of 4 are used)
		+	*put septum on top, press, tap once (do NOT mix)
		+	*keep on ice in aluminium rack
		+	*Heat 3 min at 95°C (SWATI/95-CST) to keep in single stranded form)
		+	*keep on ice in aluminium rack

	==Notes==		==Notes==

Christopher C Vanlang: /* Material */

2011-12-22T09:03:39Z

Material

←Older revision		Revision as of 09:03, 22 December 2011
Line 5:		Line 5:
	==Material==		==Material==
	* 100% ethanol (for analysis)		* 100% ethanol (for analysis)
		+	* 70% ethanol (store @ 4°C)
	* 3M sodium acetate pH 5.2 (store @ 4°C)		* 3M sodium acetate pH 5.2 (store @ 4°C)
	* 5M ammonium acetate		* 5M ammonium acetate

Christopher C Vanlang: /* Material */

2011-12-22T09:03:06Z

Material

←Older revision		Revision as of 09:03, 22 December 2011
Line 5:		Line 5:
	==Material==		==Material==
	* 100% ethanol (for analysis)		* 100% ethanol (for analysis)
-	* 3M sodium acetate pH 5.2	+	* 3M sodium acetate pH 5.2 (store @ 4°C)
	* 5M ammonium acetate		* 5M ammonium acetate
	* -20°C freezer		* -20°C freezer

Kevin W. Tosh at 18:15, 17 February 2011

2011-02-17T18:15:17Z

←Older revision		Revision as of 18:15, 17 February 2011
Line 14:		Line 14:
	[[Image:Tube placement in centrifuge.jpg\|thumb\|200px\|How to arrange tubes in the centrifuge.]]		[[Image:Tube placement in centrifuge.jpg\|thumb\|200px\|How to arrange tubes in the centrifuge.]]
	[[Image:DNA EtOH precipitation small pellet.jpg\|thumb\|200px\|Pellets can be very small and hard to see.]]		[[Image:DNA EtOH precipitation small pellet.jpg\|thumb\|200px\|Pellets can be very small and hard to see.]]
-	#Add the following to your sample:	+	#Add the following to your sample in the order they appear:
-	~~#*2-3 volumes of 100% Ethanol~~	+
	#*1/10 volume of 3M sodium acetate, pH 5.2 or 1/2 volume of 5M ammonium acetate		#*1/10 volume of 3M sodium acetate, pH 5.2 or 1/2 volume of 5M ammonium acetate
		+	#*2-3 volumes of 100% Ethanol
	#Mix and freeze overnight in -20. This step some say is unnecessary but others swear by it. If you are in a rush you can also put it in the -80 for ten minutes to a few hours. Dry ice for 10-15 minutes also works.--[[User:Etchevers\|Heather]]		#Mix and freeze overnight in -20. This step some say is unnecessary but others swear by it. If you are in a rush you can also put it in the -80 for ten minutes to a few hours. Dry ice for 10-15 minutes also works.--[[User:Etchevers\|Heather]]
	#*In general, the time you need to incubate in the freezer depends on how much nucleic acid you have, how big it is and the volume it is in. My general protocol is to freeze for 20 min to 1 hr at -80 ˚C. This seems to work well for most things, but you may want to freeze longer if you have only a small concentration of nucleic acid or if it is small in size(<15 nucleotides).--[[User:Kathmc\|Kathleen]]		#*In general, the time you need to incubate in the freezer depends on how much nucleic acid you have, how big it is and the volume it is in. My general protocol is to freeze for 20 min to 1 hr at -80 ˚C. This seems to work well for most things, but you may want to freeze longer if you have only a small concentration of nucleic acid or if it is small in size(<15 nucleotides).--[[User:Kathmc\|Kathleen]]

Kevin W. Tosh at 18:13, 17 February 2011

2011-02-17T18:13:03Z

←Older revision		Revision as of 18:13, 17 February 2011
Line 5:		Line 5:
	==Material==		==Material==
	* 100% ethanol (for analysis)		* 100% ethanol (for analysis)
-	* sodium acetate	+	* 3M sodium acetate pH 5.2
		+	* 5M ammonium acetate
	* -20°C freezer		* -20°C freezer
	* Optional: you can use linear polyacrylamide, glycogen or tRNA as precipitation carrier --[[User:Modesto Redrejo Rodriguez\|zurdo]] 03:45, 9 October 2009 (EDT)		* Optional: you can use linear polyacrylamide, glycogen or tRNA as precipitation carrier --[[User:Modesto Redrejo Rodriguez\|zurdo]] 03:45, 9 October 2009 (EDT)
Line 15:		Line 16:
	#Add the following to your sample:		#Add the following to your sample:
	#*2-3 volumes of 100% Ethanol		#*2-3 volumes of 100% Ethanol
-	#*1/10 volume of 3M sodium acetate, pH 5.2	+	#*1/10 volume of 3M sodium acetate, pH 5.2 or 1/2 volume of 5M ammonium acetate
	#Mix and freeze overnight in -20. This step some say is unnecessary but others swear by it. If you are in a rush you can also put it in the -80 for ten minutes to a few hours. Dry ice for 10-15 minutes also works.--[[User:Etchevers\|Heather]]		#Mix and freeze overnight in -20. This step some say is unnecessary but others swear by it. If you are in a rush you can also put it in the -80 for ten minutes to a few hours. Dry ice for 10-15 minutes also works.--[[User:Etchevers\|Heather]]
	#*In general, the time you need to incubate in the freezer depends on how much nucleic acid you have, how big it is and the volume it is in. My general protocol is to freeze for 20 min to 1 hr at -80 ˚C. This seems to work well for most things, but you may want to freeze longer if you have only a small concentration of nucleic acid or if it is small in size(<15 nucleotides).--[[User:Kathmc\|Kathleen]]		#*In general, the time you need to incubate in the freezer depends on how much nucleic acid you have, how big it is and the volume it is in. My general protocol is to freeze for 20 min to 1 hr at -80 ˚C. This seems to work well for most things, but you may want to freeze longer if you have only a small concentration of nucleic acid or if it is small in size(<15 nucleotides).--[[User:Kathmc\|Kathleen]]

Vaishnavi Ananth at 09:40, 19 November 2009

2009-11-19T09:40:20Z

←Older revision		Revision as of 09:40, 19 November 2009
Line 34:		Line 34:
	*[[Isopropanol Precipitation for PCR Purification]] - 2-propanol instead of ethanol		*[[Isopropanol Precipitation for PCR Purification]] - 2-propanol instead of ethanol

-	==~~BioStream~~ version==	+	==BioCoder version==
-	Following is the Ethanol precipitation of nucleic acids protocol in ~~BioStream~~, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in ~~BioStream~~ (see Source code). More information about ~~BioStream~~ can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]	+	Following is the Ethanol precipitation of nucleic acids protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]
	====Text Output====		====Text Output====
	[[Ethanol precipitation of nucleic acids protocol]]		[[Ethanol precipitation of nucleic acids protocol]]

Vaishnavi Ananth at 06:06, 23 October 2009

2009-10-23T06:06:51Z

←Older revision		Revision as of 06:06, 23 October 2009
Line 34:		Line 34:
	*[[Isopropanol Precipitation for PCR Purification]] - 2-propanol instead of ethanol		*[[Isopropanol Precipitation for PCR Purification]] - 2-propanol instead of ethanol

		+	==BioStream version==
		+	Following is the Ethanol precipitation of nucleic acids protocol in BioStream, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioStream (see Source code). More information about BioStream can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]
		+	====Text Output====
		+	[[Ethanol precipitation of nucleic acids protocol]]
		+	====Source Code====
		+	[[Ethanol precipitation of nucleic acids protocol - source code]]

	[[Category:Protocol]]		[[Category:Protocol]]