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Ethanol Precipitation
Precipitating DNA/RNA from solution to remove salts and small nucleic acid fragments. |
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- 3M Sodium Acetate buffer, pH 5.2 (store at 4°C)
- Cold 100% Ethanol (–20°C)
- Cold 70% Ethanol in sterile dH2O (–20°C)
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- 3M Sodium Acetate, pH 5.2 (store at 4°C)
- Make by dissolving 408.3 g sodium acetate trihydrate in ~800 ml H2. Titrating pH with glacial acetic acid in a hood (being careful of the fumes) to pH 5.2. And then adding water to reach final volume of 1 L.
- Cold 100% Ethanol (–20°C) Be sure to store in a flammable safe freezer!
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- Cold 70% Ethanol in sterile dH2O (–20°C) Be sure to store in a flammable safe freezer!
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- 4°C Microcentrifuge (or normal microcentrifuge in cold room).
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- Transfer DNA to a 500µl tube.
- Add one tenth volume of Sodium Acetate buffer to equalize ion concentrations.
- Add at least two volumes of cold 100% ethanol.
- Add 1-3µl linear acrylamide, mix, and let stand in –20°C freezer for at least one hour.***
- Centrifuge 14,000×g for 30 minutes at 4°C (to centrifuge we transferred reaction to a chilled 1.5µl tube).
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- Transfer DNA to a 1.7 ml Eppendorf tube. If your sample volume is < 200 µl, it can be helpful to add dH2O to reach 200 µl.
- Add a volume of Sodium Acetate equal to one tenth the sample volume. This provides the high ion concentration and right pH for the DNA to precipitate.
- Add a volume of 100% ethanol equal to 2.5 times the original sample. Use ethanol that has been pre-chilled to –20°C.
- Add 2 µl linear acrylamide (10 µg) to the sample, mix, and let stand in –20°C freezer for at least 30 minutes. In some cases longer in the freezer or overnight may improve recovery.
- Centrifuge 14,000×g for 15 minutes at 4°C.
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- Remove as much supernatant as possible with a 1ml pipet; then remove the rest with a 200µl pipet.
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- Add 200µl of cold 70% ethanol; centrifuge for 10 minutes at 4°C.
- Remove supernatant with a 200µl pipet; evaporate residual ethanol using a speed-vac or by opening the tube and leaving at room temperature or in a 37°C heat block.
- Resuspend pellet in water or a new buffer of choice to an appropriate concentration.
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- Add 200µl of cold 70% ethanol. Pipet up and down until pellet dislodges from the side of the tube. Centrifuge for 10 minutes at 4°C.
- Remove supernatant with a 200µl pipet. Evaporate residual ethanol using a speed-vac or by opening the tube and leaving at room temperature or in a 37°C heat block.
- Resuspend pellet in water or a new buffer of choice to an appropriate concentration [2].
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< | * So for a 60µl ligation reaction: 60µl DNA, 6µl Sodium acetate, 120µl 100% EtOH, & 2µl acrylamide/ |
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> | * So for a 60µl ligation reaction: 60µl DNA, 6µl Sodium acetate, 150µl 100% EtOH, and 2 µl acrylamide. |
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< | (there's an interesting typo in the Ambion protocol for this... the acrylamide stock is 5mg/ml and the protocol says you want a final concentration of 10-20 g/ml linear acrylamide... I assume they meant µg/ml... 2µl of a 5mg/ml stock seems to be the consensus elsewhere online... that would be 10µg in 188µl or ~50µg/ml, but that was fine.) |
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> | Variation: When precipitating single-stranded nucleic acid samples it may improve recovery to resuspend in 0.001% SDS to prevent sticking to the wall of the tube. |
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-- Main.LindseyWolf - 02 Dec 2011 |
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