Plant DNA Extraction

Summary

The extraction of plant DNA is a basic technique in plant molecular biology and genetic engineering research, and several methods such as CTAB method, SDS method, chlorination section method, high salt and low pH method, pectinase method, etc., have been used so far.

Principle

1、CTAB method

CTAB is a cationic decontaminant that can form complexes with nucleic acids, which will precipitate in low-salt solutions due to reduced solubility and dissociate in high-salt solutions, thus separating DNA and polysaccharides, and then precipitate DNA with ethanol to remove CTAB. polyvinylphenone (PVP) used in this method combines with polyphenols to form a complex, thus effectively preventing polyphenols from affecting the degradation of DNA. In this method, polyvinylphenone (PVP) binds to polyphenols to form a complex, thus effectively avoiding the influence of polyphenols on DNA degradation.

2. SDS method

Compared with CTAB, SDS method is more convenient and simple. In recent years, researchers have proposed the method of using SDSTris-Cl-EDTA to directly lysed cells to release DNA, and phenol/chloroform is usually used in the subsequent DNA purification process, but it is not a good choice for plant DNA purification because the use of phenol can reduce the efficiency and purity of DNA extraction. Experiments have shown that if larger centrifugation force and longer centrifugation time are used, and then chloroform-isoamyl alcohol is used instead of phenol to remove denatured proteins, it can overcome the difficulties brought about by phenol adulteration and residue to the subsequent enzyme digestion.

The most important feature of this method is that SDS is used as a decontaminant of cell membrane and denaturant of protein at the same time, and the denatured protein is removed by centrifugation instead of phenol extraction, which makes the whole operation process simple, fast and gentle, and the purified plant DNA can be obtained in about 1 h. The DNA obtained by this method can be directly digested by restriction endonuclease, and has a high purity, which is suitable for research in molecular biology. It is suitable for molecular biology research.

3、Chlorination section method

The extraction rate of chlorinated nodules is higher. The reason may be that the chlorination section not only reacts with the polysaccharide on the plant cell wall to generate ether, but also reacts with the All group of polysaccharide in the cellular fluid to destroy the sugar chain, which is conducive to the release and purification of DNA.

4、High salt and low pH method

High salt and low pH method refers to inorganic salt and isopropylene as the purification reagent DNA extraction method. Usually the inorganic salt used is potassium acetate with low pH, which is used to precipitate protein. This method is convenient and time-saving, and requires a small amount of sample.

5、Pectinase method

This is a technology that utilizes pectinase to extract the broken cells of plants.

Operation method

A simple and practical experiment for extracting plant DNA

Principle

The extraction of SDS is a basic technique in plant molecular biology and genetic engineering research, and several methods have been used so far. The most important feature of this method is that SDS is used as a decontaminant of cell membrane and denaturant of protein at the same time, and the denatured protein is removed by centrifugation instead of phenol extraction, which makes the whole operation process simple, fast and gentle, and the purified plant DNA can be obtained in about 1 h. DNA obtained by this method can be directly digested by restriction endonuclease, and has a high purity, which is suitable for molecular biology research. Research. The purpose of this experiment is to master the principle and method of DNA extraction.

Move

I. Materials and methods:

1, Plant materials: spirulina (Spirulina platensis), rice (Oryza sativa) and tobacco (Nicotianatabacum) .

2, Reagents and solutions:

(1) 20×SSC: 3 mol-L-1 sodium citrate, pH 8.0:

(2) Lysozyme, 10 μg-μl-1Rnase, 10% SDS (all Sigma products);

(3) Phenol: chloroform: isoamyl alcohol = 25:24:1

(4) TE: 10 mmol-L-1Tris-HCL, 1 mmol-L-1EDTA, pH 7.6.

3, Extraction steps:

Spirulina cells at logarithmic growth stage were collected and suspended in 5-fold volume (v/w) of 20× SSC containing 2 mg-mL-1 lysozyme and kept at room temperature for 10 min with occasional shaking to mix. Rice seedlings and tobacco shoots were suspended in 5-fold volume of 20×SSC immediately after being ground to powder in liquid nitrogen and gently mixed. The above suspension was first left on ice for 3 min, then 10% SDS was added to a final concentration of 2% and mixed well; after continuing to leave for 10 min, it was centrifuged at 11,000 × g for 15 min at 4°C. The supernatant was transferred to a new centrifuge tube and diluted with the same volume of TE, then RNase was added to a final concentration of 10 μg-ml-1 and left at room temperature for 30 min. centrifugation was performed at 11,000 × g for 15 min, and the resulting DNA precipitate was washed twice with 70% ethanol, then drained in a vacuum or naturally dried, and finally dissolved in an appropriate amount of TE.

II.Results and Discussion:

Using this method, DNA was extracted from tobacco, rice and spirulina.Because the whole process had fewer and gentler steps, which reduced the chances of the DNA being broken, the resulting DNA had a larger molecular weight, most of which was larger than that of the intact λ phage (48.5 Kb). In addition, the OD260/OD280 ratios of the DNA extracted by this method were between 1.8 and 1.9 and could be digested by restriction endonucleases, showing good purity.

When using this method, the following points must be noted. When SDS is added to 2% in a suspension of 20 x SSC, a white flocculent material appears in the solution, indicating that the protein has been denatured, which is one of the main features of this method. It removes the vast majority of the protein by centrifugation after denaturation rather than the commonly used method of multiple phenol extraction. The supernatant obtained by centrifugation needs to be diluted with TE, this is because the salt concentration in the solution is high, if it is not diluted, the RNase can not work; and in the precipitation of DNA, if ethanol will precipitate the salt in the solution, if isoamyl alcohol, it will form the interface of the two phases, and after the concentration of the salt in the solution is lowered, it solves these problems. However, increasing the volume of diluted TE does not have a very significant effect on the yield of DNA, and dilution with an equal volume of TE is sufficient.The DNA extraction solution is digested by RNase, and then extracted once more with phenol: chloroform: isoamyl alcohol to remove the remaining proteins and enzymes. This method is suitable for both bulk and trace DNA preparation. For bulk preparations, the RNase digestion and phenol:chloroform:isoamyl alcohol extraction steps can be used after the DNA has been concentrated a bit, which is easier to handle and saves RNase and phenol. Typically, 10 to 15 μg of DNA per gram of fresh tissue can be extracted using this method.

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