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interactome:trizol [2019/12/05 11:40] 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. | ||
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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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