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interactome:trizol [2019/12/05 11:40]
dan
interactome:trizol [2019/12/05 11:51]
dan
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 | RNase-free water of 0.5% SDS         ​| ​      | | RNase-free water of 0.5% SDS         ​| ​      |
 | (Optional) RNase-free glycogen ​      ​| ​      | | (Optional) RNase-free glycogen ​      ​| ​      |
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   *Always use proper microbiological aseptic techniques when working with RNA.   *Always use proper microbiological aseptic techniques when working with RNA.
   *Use RNaseZapTM RNase Decontamination Solution (Cat.no. AM9780) to remove RNase contamination from work surfaces and non-disposable items such as centrifuges and pipettes used during purification.   *Use RNaseZapTM RNase Decontamination Solution (Cat.no. AM9780) to remove RNase contamination from work surfaces and non-disposable items such as centrifuges and pipettes used during purification.
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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:
   *Remove growth media.   *Remove growth media.
   *Add 0.3–0.4 mL of TRIzolTM Reagent per 1 × 105—107 cells directly to the culture dish to lyse the cells.   *Add 0.3–0.4 mL of TRIzolTM Reagent per 1 × 105—107 cells directly to the culture dish to lyse the cells.
-  *Pipet the lysate up and down several times to homogenize. 
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-Cells grown in suspension: 
-  *Pellet the cells by centrifugation and discard the supernatant. 
-  *Add 0.75 mL of TRIzolTM Reagent per 0.25 mL of sample (5– 10 × 106 cells from animal, plant, or yeasty origin or 1 ×107 cells of bacterial origin) to the pellet. 
-**Note: Do not wash cells before addition of TRIzolTM Reagent to avoid mRNA degradation.** 
   *Pipet the lysate up and down several times to homogenize.   *Pipet the lysate up and down several times to homogenize.
  
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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.txt
  • Last modified: 2019/12/05 11:51
  • by dan