RNA extraction using trizol/tri

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== Reagents ==
== Reagents ==
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 +
* TRIzol Reagent (final concentration):
 +
 +
<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
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</pre>
 +
</small>
 +
 +
* 0.8 M sodium citrate / 1.2 M NaCl
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* isopropanol (2-propanol)
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* chloroform
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* 75% EtOH in DEPC H2O
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* RNase free water (filtered or DEPC)
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 +
<small>
 +
<pre>
 +
draw water into RNase-free glass bottles
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add diethylpyrocarbonate (DEPC) to 0.01% (v/v)
 +
let stand overnight and autoclave
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</pre>
 +
</small>
== Steps ==
== Steps ==

Revision as of 13:12, 14 February 2008

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.

Contents

Principle

Reagents

  • TRIzol Reagent (final concentration):

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

  • 0.8 M sodium citrate / 1.2 M NaCl
  • isopropanol (2-propanol)
  • chloroform
  • 75% EtOH in DEPC H2O
  • RNase free water (filtered or DEPC)

draw water into RNase-free glass bottles
add diethylpyrocarbonate (DEPC) to 0.01% (v/v)
let stand overnight and autoclave

Steps

Common mistakes

See also

External links

Reagents

Protocols

Tips

Personal tools