Trizol extraction for RNA and DNA

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(trizol vs. trizol ls note)
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==Materials==
==Materials==
*TRIzol or TRIzol LS reagent from [http://invitrogen.com Invitrogen]
*TRIzol or TRIzol LS reagent from [http://invitrogen.com Invitrogen]
 +
:*TRIzol LS is specifically created to work with liquid samples (i.e. blood and cell culture) it should be diluted when used with tissue samples.  The equivalent of 1ml of regular TRIzol is 250ul of H<sub>2</sub>O and 750ul of TRIzol LS.
*0.1M Sodium Citrate in 10% Ethanol (for 100ml 2.94g of the sodium citrate dihydrate mixed with 10ml EtOH and brought up to 100ml with H<sub>2</sub>O)
*0.1M Sodium Citrate in 10% Ethanol (for 100ml 2.94g of the sodium citrate dihydrate mixed with 10ml EtOH and brought up to 100ml with H<sub>2</sub>O)
*100% Ethanol
*100% Ethanol

Revision as of 10:03, 22 August 2007

This is a protocol for extracting both RNA and DNA from mammalian tissue. It is currently written for tissue preparation but can also be used on cell suspensions and cells grown on a monolayer.

Contents

Materials

  • TRIzol LS is specifically created to work with liquid samples (i.e. blood and cell culture) it should be diluted when used with tissue samples. The equivalent of 1ml of regular TRIzol is 250ul of H2O and 750ul of TRIzol LS.
  • 0.1M Sodium Citrate in 10% Ethanol (for 100ml 2.94g of the sodium citrate dihydrate mixed with 10ml EtOH and brought up to 100ml with H2O)
  • 100% Ethanol
  • 75% Ethanol
  • 8mM NaOH

RNA

Homoginize sample

  • Homogonize tissue in 1ml trizol per 50-100 mg of tissue without having more than 10% sample volume compared to trizol
  • Incubate sample 5' @ RT

Extract RNA

  • Add 0.2ml of chloroform per 1mL of trizol and shake for 15"
  • Spin at 12,000xg for 15' @ 4C
  • Remove top aqueous phase (SAVE bottom organic phase for DNA)

RNA Precipitation

  • Add 0.5ml of isopropanol per 1ml of trizol to the aqueous phase
  • Incubate 10' @ RT
  • Spin at 12,000xg for 10' @ 4C
  • Wash pellet with 1ml 75% EtOH per 1ml trizol then vortex
  • Spin at 12,000xg for 5' @ 4C

RNA Resuspension

  • Air dry for 5-10' and resuspend in 30-50ul RNAse free H2O

DNA

Percipitation of DNA

  • Take organic phase and carefully remove all aqueous phase
  • Add 0.4ml 100% EtOH per 1ml of trizol mix by hand
  • Incubate 2-3' @ RT
  • Spin at 2,000xg for 5' @ 4C
  • Remove sup (can be saved for protein purification)

DNA Wash

  • Wash pellet 2x with 0.1M Sodium Citrate 10% EtOH 1ml per 1ml of trizol
  • At each wash incubate in wash solution for 30' @ RT
  • Spin at 2,000xg for 5' @ 4C
  • Resuspend in 1.5-2.0ml 75% EtOH and incubate for 10-20' @ RT mix periodically
  • Spin at 2,000xg for 5' @ 4C

Redissolve DNA

  • Redissolve pellet in 300-600ul of 8mM NaOH per 50-70mg of tissue
  • Spin at 12,000xg for 10 minutes to remove cell detritus
  • Save supernatant with DNA
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