Jakob Suckale: lead section

2009-02-22T12:47:05Z

lead section

←Older revision		Revision as of 12:47, 22 February 2009
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-	=== ~~Mixing your own "TRI" reagent~~ ===	+	RNA extraction with guanidinium-acid-phenol reagents 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] is generally considered the extraction method that gives the best quality RNA. If you prefer using ready-made reagents like TRIzol (Invitrogen product name) or the equivalent TRI (Sigma-Aldrich product name) refer to this protocol page: [[RNA extraction using trizol/tri]].

-	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:	+	== 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		* 4 M guanidinium thiocyanate
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	(store <1 month at RT)		(store <1 month at RT)

-	=== Using Solution D ===	+	== Using Solution D ==

	Assuming you start with 10 million cells or 100 mg of tissue (keep cool when possible):		Assuming you start with 10 million cells or 100 mg of tissue (keep cool when possible):

Jakob Suckale: moved from trizol page

2009-02-22T12:39:49Z

moved from trizol page

New page

=== 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

RNA extraction using self-made guanidinium-acid-phenol reagents - Revision history

Jakob Suckale: lead section

Jakob Suckale: moved from trizol page