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interactome:trizol [2019/12/05 11:50]
dan
interactome:trizol [2019/12/05 11:51] (current)
dan
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   *Lyse and homogenize samples in TRIzolTM Reagent according to your starting material.   *Lyse and homogenize samples in TRIzolTM Reagent according to your starting material.
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-Tissues: 
-  *Add 1 mL of TRIzolTM Reagent per 50–100 mg of tissue to the sample and homogenize using a homogenizer. ​ 
  
 Cell grown in monolayer: Cell grown in monolayer:
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     STOPPING POINT     STOPPING POINT
     Samples can be stored at 4°C overnight or at – 20°C for up to a year.     Samples can be stored at 4°C overnight or at – 20°C for up to a year.
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   *(Optional) If samples have a high fat content, centrifuge the lysate for 5 minutes at 12,000 × g at 4–10°C, then transfer the clear supernatant to a new tube.   *(Optional) If samples have a high fat content, centrifuge the lysate for 5 minutes at 12,000 × g at 4–10°C, then transfer the clear supernatant to a new tube.
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   *Proceed directly to “Isolate RNA“ ​   *Proceed directly to “Isolate RNA“ ​
  
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 ==2  Isolate RNA== ==2  Isolate RNA==
  
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   *Total RNA precipitate forms a white gel-like pellet at the bottom of the tube.   *Total RNA precipitate forms a white gel-like pellet at the bottom of the tube.
   *Discard the supernatant with a micropipettor.   *Discard the supernatant with a micropipettor.
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 Wash the RNA Wash the RNA
   *Resuspend the pellet in 1 mL of 75% ethanol per 1 mL of TRIzolTM Reagent used for lysis.   *Resuspend the pellet in 1 mL of 75% ethanol per 1 mL of TRIzolTM Reagent used for lysis.
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     IMPORTANT! ​     IMPORTANT! ​
     Do not dry the pellet by vacuum centrifuge. Do not let the RNA pellet dry, to ensure total solubilization of the RNA. Partially dissolved RNA samples have an A230/280 ratio <1.6.     Do not dry the pellet by vacuum centrifuge. Do not let the RNA pellet dry, to ensure total solubilization of the RNA. Partially dissolved RNA samples have an A230/280 ratio <1.6.
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 Solubilize the RNA Solubilize the RNA
   *Resuspend the pellet in 20–50 μL of RNase-free water, 0.1 mM EDTA, or 0.5% SDS solution by pipetting up and down.   *Resuspend the pellet in 20–50 μL of RNase-free water, 0.1 mM EDTA, or 0.5% SDS solution by pipetting up and down.
  • interactome/trizol.1575514231.txt.gz
  • Last modified: 2019/12/05 11:50
  • by dan