RNA extraction using trizol/tri - Revision history

Jakob Suckale: categories

Jakob Suckale — Wed, 17 Jun 2009 17:00:21 GMT

Jakob Suckale: /* External links */

Jakob Suckale — Wed, 01 Apr 2009 15:10:23 GMT

External links

←Older revision		Revision as of 15:10, 1 April 2009
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	* [http://www.animal.ufl.edu/hansen/protocols/RNA_extraction.htm RNA extraction with TRIZOL] (Uni Florida)		* [http://www.animal.ufl.edu/hansen/protocols/RNA_extraction.htm RNA extraction with TRIZOL] (Uni Florida)
	* [http://www.utm.utoronto.ca/~w3flyma/protocols/Trizol%20Troubleshooting.pdf Troubleshooting guide for TRIZOL extraction] (Uni Toronto)		* [http://www.utm.utoronto.ca/~w3flyma/protocols/Trizol%20Troubleshooting.pdf Troubleshooting guide for TRIZOL extraction] (Uni Toronto)
		+	* [http://en.wikipedia.org/wiki/Phenol-chloroform_extraction Phenol-chloroform extraction of nucleic acids at Wikipedia]

	Tips		Tips
	* [http://www.ambion.com/techlib/tn/94/949.html Ambion tips on RNA extraction]		* [http://www.ambion.com/techlib/tn/94/949.html Ambion tips on RNA extraction]

Jakob Suckale: /* External links */ link updated

Jakob Suckale — Wed, 01 Apr 2009 15:05:54 GMT

External links: link updated

←Older revision		Revision as of 15:05, 1 April 2009
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	== External links ==		== External links ==
	Reagents		Reagents
-	* [http://~~www~~.invitrogen.com/~~content.cfm~~?~~pageid~~=~~469~~ TRIZOL reagent] (Invitrogen), [http://en.wikipedia.org/wiki/TRIzol TRIZOL on wikipedia]	+	* [http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&productID=15596026 TRIZOL reagent] (Invitrogen), [http://en.wikipedia.org/wiki/TRIzol TRIZOL on wikipedia]
	* [http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Molecular_Biology/DNA_and_RNA_Purification/Tri_Reagent_RNA_Isolation_Reagent.html TRI reagent] (Sigmal Aldrich)		* [http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Molecular_Biology/DNA_and_RNA_Purification/Tri_Reagent_RNA_Isolation_Reagent.html TRI reagent] (Sigmal Aldrich)

Jakob Suckale: /* Reagents */ removed details and instead placed link to more extensive DEPC page

Jakob Suckale — Sun, 22 Feb 2009 12:41:58 GMT

Reagents: removed details and instead placed link to more extensive DEPC page

←Older revision		Revision as of 12:41, 22 February 2009
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	* chloroform		* chloroform
	* 75% EtOH in DEPC H2O		* 75% EtOH in DEPC H2O
-	* RNase free water (filtered or DEPC)	+	* RNase free water (filtered or [[DEPC]])
-		+
-	~~<small>~~	+
-	~~<pre>~~	+
-	~~draw water into RNase-free glass bottles~~	+
-	~~add diethylpyrocarbonate (DEPC) to 0.01% (v/v)~~	+
-	~~let stand overnight and autoclave~~	+
-	~~</pre>~~	+
-	~~</small>~~	+

	== Steps ==		== Steps ==

Jakob Suckale: /* Reagents */ moved to separate protocol page to keep the page shorter

Jakob Suckale — Sun, 22 Feb 2009 12:40:51 GMT

Reagents: moved to separate protocol page to keep the page shorter

←Older revision		Revision as of 12:40, 22 February 2009
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	== Reagents ==		== Reagents ==

-	* TRIzol or TRI reagent	+	* TRIzol or TRI reagent
		+	: If you want to make your own reagents, see here [[RNA extraction using self-made guanidinium-acid-phenol reagents]]
	* 0.8 M sodium citrate / 1.2 M NaCl		* 0.8 M sodium citrate / 1.2 M NaCl
	* isopropanol (2-propanol)		* isopropanol (2-propanol)
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	</pre>		</pre>
	</small>		</small>
-
-	~~=== Mixing your own "TRI" reagent ===~~
-
-	So called Solution D (based on Chomczynski and Sacchi [[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=2440339 1987]/[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=17406285 2006]) is:
-
-	* 4 M guanidinium thiocyanate
-	* 25 mM sodium citrate
-	* pH 7.0
-	* 0.5% (wt/vol) N-laurosylsarcosine (Sarkosyl)
-	* 0.1 M 2-mercaptoethanol
-
-	~~Prepare stock with:~~
-	* dissolving 250 g guanidinium thiocyanate in 293 ml water at 65 °C
-	* add 17.6 ml of 0.75 M sodium citrate, pH 7.0
-	* 26.4 ml of 10% (wt/vol) N-laurosylsarcosine
-	~~(stored <3 months at room temperature)~~
-
-	~~Working solution from stock:~~
-	* add 0.36 ml of 98% 2-mercaptoethanol to 50 ml of stock solution
-	~~(store <1 month at RT)~~
-
-	~~=== Using Solution D ===~~
-
-	~~Assuming you start with 10 million cells or 100 mg of tissue (keep cool when possible):~~
-	* add 1 ml solution D (don't linger on this step)
-	* transfer to tubes
-	* add 0.1 ml of 2 M sodium acetate, pH 4.0, and invert tube to mix
-	* add 1 ml water-saturated phenol (never buffered phenol) and invert tube
-	* add 0.2 ml of chloroform/isoamyl alcohol (49:1) and shake vigorously for 10 sec
-	* centrifuge 20min 10000G 4ºC
-	* transfer top aqueous phase into new tube
-	* precipitate

	== Steps ==		== Steps ==

Jakob Suckale: /* Reagents */ using solution D

Jakob Suckale — Sun, 22 Feb 2009 12:34:41 GMT

Reagents: using solution D

←Older revision		Revision as of 12:34, 22 February 2009
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	* add 0.36 ml of 98% 2-mercaptoethanol to 50 ml of stock solution		* add 0.36 ml of 98% 2-mercaptoethanol to 50 ml of stock solution
	(store <1 month at RT)		(store <1 month at RT)
		+
		+	=== Using Solution D ===
		+
		+	Assuming you start with 10 million cells or 100 mg of tissue (keep cool when possible):
		+	* add 1 ml solution D (don't linger on this step)
		+	* transfer to tubes
		+	* add 0.1 ml of 2 M sodium acetate, pH 4.0, and invert tube to mix
		+	* add 1 ml water-saturated phenol (never buffered phenol) and invert tube
		+	* add 0.2 ml of chloroform/isoamyl alcohol (49:1) and shake vigorously for 10 sec
		+	* centrifuge 20min 10000G 4ºC
		+	* transfer top aqueous phase into new tube
		+	* precipitate

	== Steps ==		== Steps ==

Jakob Suckale: /* Reagents */ mixing your own

Jakob Suckale — Sun, 22 Feb 2009 12:27:31 GMT

Reagents: mixing your own

←Older revision		Revision as of 12:27, 22 February 2009
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	== Reagents ==		== Reagents ==

-	* TRIzol ~~Reagent (final concentration):~~	+	* TRIzol or TRI reagent
-		+
-	~~<small>~~	+
-	~~<pre>~~	+
-	~~Phenol in saturated buffer (38 %) -- 380 ml/liter~~	+
-	~~Guanidine thiocyanate (0.8 M) -- 118.16 g~~	+
-	~~Ammonium thiocyanate (0.4 M) -- 76.12 g~~	+
-	~~Sodium acetate, pH5 (0.1 M) -- 33.4 ml of 3M stock~~	+
-	~~Glycerol -- 50 ml~~	+
-	~~H2O to 1.0 liter~~	+
-	~~</pre>~~	+
-	~~</small>~~	+
-		+
	* 0.8 M sodium citrate / 1.2 M NaCl		* 0.8 M sodium citrate / 1.2 M NaCl
	* isopropanol (2-propanol)		* isopropanol (2-propanol)
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	</pre>		</pre>
	</small>		</small>
		+
		+	=== Mixing your own "TRI" reagent ===
		+
		+	So called Solution D (based on Chomczynski and Sacchi [[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=2440339 1987]/[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=17406285 2006]) is:
		+
		+	* 4 M guanidinium thiocyanate
		+	* 25 mM sodium citrate
		+	* pH 7.0
		+	* 0.5% (wt/vol) N-laurosylsarcosine (Sarkosyl)
		+	* 0.1 M 2-mercaptoethanol
		+
		+	Prepare stock with:
		+	* dissolving 250 g guanidinium thiocyanate in 293 ml water at 65 °C
		+	* add 17.6 ml of 0.75 M sodium citrate, pH 7.0
		+	* 26.4 ml of 10% (wt/vol) N-laurosylsarcosine
		+	(stored <3 months at room temperature)
		+
		+	Working solution from stock:
		+	* add 0.36 ml of 98% 2-mercaptoethanol to 50 ml of stock solution
		+	(store <1 month at RT)

	== Steps ==		== Steps ==

Jakob Suckale: statement on TRI(zol) quality

Jakob Suckale — Sun, 22 Feb 2009 12:07:09 GMT

statement on TRI(zol) quality

←Older revision		Revision as of 12:07, 22 February 2009
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	[[Image:Trizol phases.png\|thumbnail\|300px\|right]]		[[Image:Trizol phases.png\|thumbnail\|300px\|right]]

-	RNA extraction with TRIzol (Invitrogen product name) or the equivalent TRI (Sigma-Aldrich product name) is a common method of total [[RNA extraction]] from cells based on the research of Chomczynski P, Sacchi N. 1987 [http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=2440339] and reviewed by the authors again in 2006 [http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=17406285]. It takes slightly longer than column-based methods like RNAeasy but it has higher capacity and can yield more RNA.	+	RNA extraction with TRIzol (Invitrogen product name) or the equivalent TRI (Sigma-Aldrich product name) is a common method of total [[RNA extraction]] from cells based on the research of Chomczynski P, Sacchi N. 1987 [http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=2440339] and reviewed by the authors again in 2006 [http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=17406285]. It takes slightly longer than column-based methods like RNAeasy but it has higher capacity and can yield more RNA. Along with chaotropic lysis buffers it is generally considered the method that gives the best quality RNA.

	== Principle ==		== Principle ==

Jakob Suckale: referenes

Jakob Suckale — Sun, 22 Feb 2009 12:05:38 GMT

referenes

←Older revision		Revision as of 12:05, 22 February 2009
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	[[Image:Trizol phases.png\|thumbnail\|300px\|right]]		[[Image:Trizol phases.png\|thumbnail\|300px\|right]]

-	RNA extraction with ~~TRIZOL~~ (Invitrogen product name) or the equivalent TRI (Sigma-Aldrich product name) is a common method of total [[RNA extraction]] from cells. It takes slightly longer than column-based methods like RNAeasy but it has higher capacity and can yield more RNA.	+	RNA extraction with TRIzol (Invitrogen product name) or the equivalent TRI (Sigma-Aldrich product name) is a common method of total [[RNA extraction]] from cells based on the research of Chomczynski P, Sacchi N. 1987 [http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=2440339] and reviewed by the authors again in 2006 [http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=17406285]. It takes slightly longer than column-based methods like RNAeasy but it has higher capacity and can yield more RNA.

	== Principle ==		== Principle ==

Jakob Suckale: /* RNA wash */

Jakob Suckale — Tue, 06 May 2008 12:39:19 GMT

RNA wash

←Older revision		Revision as of 12:39, 6 May 2008
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	[[Image:Optional step.png]] optional add RNase inhibitor		[[Image:Optional step.png]] optional add RNase inhibitor
		+
	[[Image:Optional step.png]] incubate at 55-60 C° for 10 min if hard to redissolve		[[Image:Optional step.png]] incubate at 55-60 C° for 10 min if hard to redissolve

←Older revision		Revision as of 17:00, 17 June 2009
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	Tips		Tips
	* [http://www.ambion.com/techlib/tn/94/949.html Ambion tips on RNA extraction]		* [http://www.ambion.com/techlib/tn/94/949.html Ambion tips on RNA extraction]
		+
		+	[[Category:Protocol]]
		+	[[Category:RNA]]
		+	[[Category:In vitro]]