Step-by-step protocol for DNA precipitation using isopropanol
Equipment and reagents required
Equipment
- Centrifuge capable of 10,000–15,000 x g with rotor for the appropriate centrifuge tubes
- Centrifuge tubes
- Pipettes and pipette tips
Reagents
- Sodium acetate or ammonium acetate
- Isopropanol (100%)
- Ethanol (70%)
- DNA resuspension buffer
DNA precipitation
- Adjust the salt concentration if necessary, for example, with sodium acetate (0.3 M, pH 5.2, final concentration) or ammonium acetate (2.0–2.5 M, final concentration).
- Add 0.6–0.7 volumes of room-temperature isopropanol to the DNA solution and mix well.
Tip: Use all solutions at room temperature to minimize co-precipitation of salt.
Tip: Do not use polycarbonate tubes for precipitation as polycarbonate is not resistant to isopropanol.
- Centrifuge the sample immediately at 10,000–15,000 x g for 15–30 min at 4°C.
Tip: Centrifugation should be carried out at 4°C to prevent overheating of the sample. (When precipitating from small volumes, centrifugation may be carried out at room temperature.)
Tip: Genomic DNA can alternatively be precipitated by spooling the DNA using a glass rod following addition of isopropanol. The spooled DNA should be transferred immediately to a microfuge tube containing an appropriate buffer and redissolved (see step 9).
- Carefully decant the supernatant without disturbing the pellet.
Tip: Marking the outside of the tube before centrifugation allows the pellet to be more easily located. Pellets from isopropanol precipitation have a glassy appearance and may be more difficult to see than the fluffy salt-containing pellets resulting from ethanol precipitation.
Tip: Care should be taken when removing the supernatant as pellets from isopropanol precipitation are more loosely attached to the side of the tube.
Tip: Carefully tip the tube with the pellet on the upper side to avoid dislodging the pellet.
Tip: For valuable samples, the supernatant can be retained until recovery of the precipitated DNA has been verified.
Wash DNA pellet
- Wash the DNA pellet by adding 1–10 ml (depending on the size of the preparation) of room-temperature 70% ethanol. This removes co-precipitated salt and replaces the isopropanol with the more volatile ethanol, making the DNA easier to redissolve.
- Centrifuge at 10,000–15,000 x g for 5–15 min at 4°C.
Tip: Centrifuge the tube in the same orientation as previously to recover the DNA into a compact pellet. - Carefully decant the supernatant without disturbing the pellet.
- Air-dry the pellet for 5–20 min (depending on the size of the pellet).
Tip: Do not overdry the pellet (e.g., by using a vacuum evaporator) as this will make DNA, especially high-molecular-weight DNA, difficult to redissolve.
Resuspend DNA pellet
- Resuspend the DNA in a suitable buffer.
Tip: Choose an appropriate volume of buffer according to the expected DNA yield and the desired final DNA concentration.
Tip: Use a buffer with a pH of 7.5–8.0, as DNA does not dissolve easily in acidic buffers. (If using water, check pH.)
Tip: Redissolve by rinsing the walls to recover all the DNA, especially if glass tubes have been used. To avoid shearing the DNA, do not pipet or vortex.
Tip: High-molecular-weight DNA, such as genomic DNA, should be redissolved very gently to avoid shearing, e.g., at room temperature overnight or at 55°C for 1–2 h with gentle agitation.
Troubleshooting common problems in isopropanol DNA precipitation
| Problem | Solution |
|---|---|
| Little or no DNA after isopropanol precipitation | |
| DNA failed to precipitate | Ensure that the precipitate is centrifuged at 10,000–15,000 x g for 15–30 min at 4°C. Recover DNA by centrifuging for longer and at higher speeds. Try another isopropanol batch. |
| DNA pellet was lost | Isopropanol pellets are glassy and may be difficult to see. Mark the outside of the tube before centrifugation. Isopropanol pellets may also be loosely attached to the side of the tube, so pour supernatant off gently. |
| DNA was poorly redissolved | Check that DNA is completely redissolved. Be sure to wash any DNA off the walls, particularly if glass tubes and a fixed-angle rotor are used. Up to half of the total DNA may be smeared on the walls. Alternatively, a swinging bucket rotor can be used to ensure that the pellet is located at the bottom of the tube. |
| Incorrect isopropanol concentration | Use the correct concentration of isopropanol, i.e., 0.6–0.7 volumes. |
| Sample was not thoroughly mixed with isopropanol | Complete mixing with isopropanol is vital to precipitate the DNA. |
| DNA pellet was difficult to resuspend, or was loose and discarded with the supernatant |
Centrifugation speed was incorrect. Ensure that the precipitate is centrifuged at 10,000–15,000 x g for 15–30 min at 4°C. |
| No DNA pellet visible | Centrifugation speed after addition of isopropanol was too low. Ensure that the precipitate is centrifuged at 10,000–15,000 x g for 15–30 min at 4°C to pellet the DNA. |
| Pellet is difficult to redissolve after DNA isopropanol precipitation | |
| Pellet was overdried | Air-dry pellet instead of using a vacuum, especially if the DNA is of high molecular weight. Redissolve DNA by warming the solution slightly, and allowing more time for redissolving. |
| Residual isopropanol in pellet | Ensure that pellets are washed with 70% ethanol to remove traces of isopropanol. Redissolve DNA by warming the solution slightly, and allowing more time for redissolving. Increase volume of buffer used for redissolving if necessary. |
| Too much salt in pellet | Ensure that isopropanol is at room temperature for precipitation, and wash the pellet twice with room temperature 70% ethanol. Recover DNA by increasing the volume of buffer used for redissolving. |
| Buffer pH was too low | Ensure that the pH of the buffer used for redissolving is ≥8.0, since DNA does not dissolve well in acidic solutions. |
What are the advantages and disadvantages of isopropanol DNA precipitation?
Role of sodium acetate in DNA precipitation
Sodium acetate provides sodium ions that neutralize DNA’s negative charge, making it less soluble in water. This allows DNA molecules to aggregate and efficiently precipitate when isopropanol is added. In the presence of sodium acetate and isopropanol, the DNA molecules form visible pellets after centrifugation.
Ethanol versus isopropanol DNA precipitation: What’s the difference?
Use ethanol for:
- Small sample volumes
- High DNA concentrations
- Small DNA fragments
- Chilled storage of DNA for extended periods
Use isopropanol for:
- Large sample volumes
- Low DNA concentrations
- Larger DNA fragments
- Faster DNA precipitation
- Room temperature precipitation
Post-precipitation DNA quantification and storage
After precipitation, the DNA needs to be quantified before use in downstream applications. Quantification of DNA can be performed using a variety of methods, including spectrophotometry. Spectrophotometric measurement of DNA concentration measures the absorbance at 260 nm (A260). Pure DNA has an A260/A280 ratio of 1.8–2.0 in 10 mM Tris·Cl, pH 8.5.
Correct storage of DNA is crucial to prevent degradation and to ensure reproducible results in downstream applications.
