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interactome:trizol [2019/12/05 11:39]
dan
interactome:trizol [2019/12/05 11:50]
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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   *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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 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.
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     IMPORTANT! ​     IMPORTANT! ​
     Do not dissolve the RNA in 0.5% SDS if the RNA is to be used in subsequent enzymatic reactions.     Do not dissolve the RNA in 0.5% SDS if the RNA is to be used in subsequent enzymatic reactions.
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   *Incubate in a water bath or heat block set at 55–60°C for 10–15 minutes. ​   *Incubate in a water bath or heat block set at 55–60°C for 10–15 minutes. ​
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 Proceed to downstream applications,​ or store the RNA at –70°C. Proceed to downstream applications,​ or store the RNA at –70°C.
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 + --- //​[[daney@sfc.keio.ac.jp|Dan Yamamoto-Evans]] 2019/12/05 11:39//
  • interactome/trizol.txt
  • Last modified: 2019/12/05 11:51
  • by dan