How Isopropanol Precipitates DNA: Principles and Applications

DNA extraction and purification are crucial steps in molecular biology experiments. In order to extract pure DNA from cells or tissues, different chemical reagents and procedures are usually required. Isopropanol precipitation is a common and effective DNA precipitation technique, which is widely used in various biological experiments. How does isopropanol precipitate DNA? This article will introduce its principle, operation steps and application in detail.

Principle of DNA Precipitation with Isopropanol

DNA is made up of long chains of DNA molecules, usually dissolved in water or a saline solution. When isopropanol is added, the polarity in the solution is greatly changed. The non-polar nature of isopropanol causes the DNA molecule to gradually lose hydration, resulting in reduced solubility of DNA in solution. At this time, the DNA molecules will aggregate and precipitate out.

Isopropanol plays two key roles in the process of precipitating DNA:

1. depolarization effect: The addition of isopropanol reduces the polarity of the water, so that the DNA molecules cannot be kept in solution, thereby promoting its precipitation.
2. With the help of salt ions when extracting DNA, salts (such as sodium chloride or ammonium chloride) are usually added. The role of salt is to neutralize the negative charge on the DNA molecule, reduce the electrostatic repulsion between molecules, and make DNA easier to aggregate and precipitate.

Procedure: How to Precipitate DNA with Isopropanol

the experimental procedure for DNA precipitation using isopropanol is relatively simple, but requires precise control of solution conditions. The following is the standard operating procedure:

1. DNA Solution Preparation DNA is first extracted from cells or tissues, usually using a lysis solution (such as Tris, EDTA, NaCl, etc.) to disrupt the cell membrane and release the DNA. Then, cell debris was removed by centrifugation to obtain a supernatant.
2. Add salt solution: In order to enhance the precipitation effect of DNA, add an appropriate amount of salt solution to the supernatant. Commonly used salts are sodium chloride (NaCl) or ammonium chloride (NH4Cl), which help neutralize the negative charge of DNA molecules and improve the efficiency of DNA precipitation.
3. Add isopropyl alcohol: Add isopropanol equal to or close to the volume of the solution to the DNA solution and mix gently. At this point, the DNA molecules will begin to precipitate.
4. DNA pellet collected by centrifugation: Put the mixed solution in a centrifuge and centrifuge at low speed, usually 8000-12000 r/min minutes, and centrifuge for about 10-15 minutes. At this point, a DNA precipitate will appear at the bottom of the centrifuge tube.
5. Cleaning and dissolution: Carefully remove the supernatant and wash the DNA pellet with 70% ethanol to remove residual salts and other impurities. Finally, the pellet is dissolved in an appropriate solution (e. g., TE buffer or water) for subsequent analysis.

Advantages and Limitations of DNA Precipitation with Isopropanol

advantage

1. simple and easy: The isopropanol precipitation method is simple to operate and does not require complex instruments and equipment.
2. Efficiency: Under the condition of appropriate salt concentration, isopropanol can effectively precipitate DNA with high purity, which is suitable for most DNA extraction experiments.
3. low cost: Compared with other DNA purification methods, the use cost of isopropanol is lower, and it is suitable for small and medium-sized laboratories.

Limitations

1. may lead to contamination: If the experiment is not performed properly, it may cause the degradation of DNA or bring in other impurities.
2. Not applicable to all samples: The DNA in some special samples may be difficult to recover due to excessive precipitation, or the precipitation effect may be poor due to other insoluble substances in the sample.

Common problems in experiments: Causes of failure in isopropanol precipitation of DNA

although isopropanol precipitation is very common in DNA extraction, it is possible that precipitation fails. Common causes include:

1. inappropriate salt concentration: Too low salt concentration may lead to incomplete precipitation of DNA molecules; too high salt concentration may lead to incomplete precipitation of DNA with too much salt.
2. Incorrect proportion of isopropyl alcohol incorrect ratio of isopropanol volume to solution may affect DNA precipitation. In general, the volume of isopropanol should be 1 times the volume of the DNA solution.
3. Insufficient centrifugal speed: If the centrifugation speed is too low or the time is too short, the DNA may not be completely precipitated. It is necessary to ensure that the centrifugation conditions are sufficiently stringent to ensure efficient DNA recovery.

Summary

extraction of DNA by isopropanol precipitation is a simple and efficient method. Understanding the principle of how isopropanol precipitates DNA, mastering the correct operation steps, and overcoming possible experimental problems can help researchers better carry out DNA extraction and purification. Although this method is widely used in many experiments, it also needs to adjust the operation details according to the specific experimental requirements to obtain high-quality DNA samples.