Ethanol Precipitation

Precipitating DNA/RNA from solution to remove salts and small nucleic acid fragments.

Materials

• 3M Sodium Acetate, pH 5.2 (store at 4°C)
  • Make by dissolving 408.3 g sodium acetate trihydrate in ~800 ml H₂O. 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!
• Cold 70% Ethanol in sterile dH₂O (–20°C) Be sure to store in a flammable safe freezer!
• DNA sample

• 4°C Microcentrifuge (or normal microcentrifuge in cold room).

Procedure

1. 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.
2. 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.
3. Add a volume of 100% ethanol equal to 2.5 times the original sample. Use ethanol that has been pre-chilled to –20°C.

1. Centrifuge 14,000×g for 15 minutes at 4°C.
2. Remove as much supernatant as possible with a 1ml pipet; then remove the rest with a 200µl pipet.
3. 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.
4. 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.
5. Resuspend pellet in water or a new buffer of choice to an appropriate concentration [2].

* So for a 60µl ligation reaction: 60µl DNA, 6µl Sodium acetate, 150µl 100% EtOH, and 2 µl acrylamide.

Variation: To prevent single-stranded nucleic acids from sticking to the tube's side walls one can resuspend in 0.001% SDS + TE or 0.001% SDS + Nanopure H₂O.

Ethanol Precipitation

Precipitating DNA/RNA from solution to remove salts and small nucleic acid fragments.

Materials

• 3M Sodium Acetate, pH 5.2 (store at 4°C)

• Cold 100% Ethanol (–20°C) Be sure to store in a flammable safe freezer!
• Cold 70% Ethanol in sterile dH₂O (–20°C) Be sure to store in a flammable safe freezer!
• DNA sample
• Linear acrylamide (5 mg/ml) (Ambion Catalog #AM9520) (function of polyacrylamide: carrier that increases efficiency of DNA precipitation)
• 4°C Microcentrifuge (or normal microcentrifuge in cold room).

Procedure

1. 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.
2. 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.
3. Add a volume of 100% ethanol equal to 2.5 times the original sample. Use ethanol that has been pre-chilled to –20°C.
4. 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.
5. Centrifuge 14,000×g for 15 minutes at 4°C.
6. Remove as much supernatant as possible with a 1ml pipet; then remove the rest with a 200µl pipet.
7. 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.
8. 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.
9. Resuspend pellet in water or a new buffer of choice to an appropriate concentration [2].

* So for a 60µl ligation reaction: 60µl DNA, 6µl Sodium acetate, 150µl 100% EtOH, and 2 µl acrylamide.

Variation: To prevent single-stranded nucleic acids from sticking to the tube's side walls one can resuspend in 0.001% SDS + TE or 0.001% SDS + Nanopure H₂O.

Ethanol Precipitation

Precipitating DNA/RNA from solution to remove salts and small nucleic acid fragments.

Materials

• 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!
• Cold 70% Ethanol in sterile dH₂O (–20°C) Be sure to store in a flammable safe freezer!
• DNA sample
• Linear acrylamide (5 mg/ml) (Ambion Catalog #AM9520) (function of polyacrylamide: carrier that increases efficiency of DNA precipitation)
• 4°C Microcentrifuge (or normal microcentrifuge in cold room).

Procedure

1. 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.
2. 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.
3. Add a volume of 100% ethanol equal to 2.5 times the original sample. Use ethanol that has been pre-chilled to –20°C.
4. 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.
5. Centrifuge 14,000×g for 15 minutes at 4°C.
6. Remove as much supernatant as possible with a 1ml pipet; then remove the rest with a 200µl pipet.
7. 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.
8. 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.
9. Resuspend pellet in water or a new buffer of choice to an appropriate concentration [2].

* So for a 60µl ligation reaction: 60µl DNA, 6µl Sodium acetate, 150µl 100% EtOH, and 2 µl acrylamide.

Ethanol Precipitation

Precipitating DNA/RNA from solution to remove salts and small nucleic acid fragments.

Materials

• 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!
• Cold 70% Ethanol in sterile dH₂O (–20°C) Be sure to store in a flammable safe freezer!
• DNA sample

• 4°C Microcentrifuge (or normal microcentrifuge in cold room).

Procedure

1. 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.
2. 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.
3. Add a volume of 100% ethanol equal to 2.5 times the original sample. Use ethanol that has been pre-chilled to –20°C.
4. 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.
5. Centrifuge 14,000×g for 15 minutes at 4°C.
6. Remove as much supernatant as possible with a 1ml pipet; then remove the rest with a 200µl pipet.
7. 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.
8. 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.
9. Resuspend pellet in water or a new buffer of choice to an appropriate concentration [2].

* So for a 60µl ligation reaction: 60µl DNA, 6µl Sodium acetate, 150µl 100% EtOH, and 2 µl acrylamide.

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.

Ethanol Precipitation

Precipitating DNA/RNA from solution to remove salts and small nucleic acid fragments.

• DNA sample

• 4°C Microcentrifuge (or normal microcentrifuge in cold room).

1. Remove as much supernatant as possible with a 1ml pipet; then remove the rest with a 200µl pipet.

• 3M Sodium Acetate buffer, pH 5.2 (store at 4°C)

• DNA sample
• Linear acrylamide
• 4°C Microcentrifuge (or normal microcentrifuge in cold room).

Procedure

1. Add one tenth volume of Sodium Acetate buffer to equalize ion concentrations.
2. Add at least two volumes of cold 100% ethanol.

-- Main.LindseyWolf - 02 Dec 2011

Ethanol_precipitation

Precipitating out salts from DNA samples

Materials

• 3M Sodium Acetate buffer, pH 5.2 (store at 4°C)
• Cold 100% Ethanol (-20°C)
• Cold 70% Ethanol in sterile dH2O (-20°C)
• DNA sample

1. Add one tenth volume of Sodium Acetate buffer to equalize ion concentrations.

1. Add 200ul of cold 70% ethanol; centrifuge for 10 minutes at 4°C.
2. Remove supernatant with a 200ul pipet; evaporate remaining ethanol in a 37°C water bath or heat block.

-- Main.LindseyWolf - 02 Dec 2011

Ethanol_precipitation

Precipitating out salts from DNA samples

Materials

• 3M Sodium Acetate buffer, pH 5.2 (store at 4°C)
• Cold 100% Ethanol (-20°C)
• Cold 70% Ethanol in sterile dH2O (-20°C)
• DNA sample
• 4°C Microcentrifuge (or normal microcentrifuge in cold room). All centrifuges should be on "soft" (no brake) setting.

Procedure

1. Transfer DNA to a container where it fills one fourth the total volume (a 500ul tube should have no more that 125ul of DNA solution, for example.
2. Add one tenth volume of Sodium Acetate buffer to equalize ion concentrations.
3. Add at least two volumes of cold 100% ethanol; let stand in -20°C freezer for at least one hour.

1. Remove supernatant with a 200ul pipet; evaporate remaining ethanol in a 37°C water bath or heat block.
2. Resuspend pellet in 50ul of water or TE buffer.

-- Main.LindseyWolf - 02 Dec 2011

Ethanol_precipitation

Precipitating out salts from DNA samples

Materials

• 3M Sodium Acetate buffer, pH 5.2 (store at 4°C)
• Cold 100% Ethanol (-20°C)
• Cold 70% Ethanol in sterile dH2O (-20°C)
• DNA sample
• 4°C Microcentrifuge (or normal microcentrifuge in cold room). All centrifuges should be on "soft" (no brake) setting.

Procedure

1. Transfer DNA to a container where it fills one fourth the total volume (a 500ul tube should have no more that 125ul of DNA solution, for example.
2. Add one tenth volume of Sodium Acetate buffer to equalize ion concentrations.
3. Add at least two volumes of cold 100% ethanol; let stand in -20°C freezer for at least one hour.
4. Remove as much supernatant as possible with a 1ml micropipet; reventrifuge, then remove the rest with a 200ul pipet.
5. Add 200i; of cold 70% ethanol; centrifuge for 5 minutes at 4°C.
6. Remove supernatant with a 200ul pipet; evaporate remaining ethanol in a 37°C water bath or heat block.
7. Resuspend pellet in 50ul of water or TE buffer.

-- Main.LindseyWolf - 02 Dec 2011