The separation of high-quality DNA is a prerequisite for molecular research. Compared with animals, maintaining the yield and quality of DNA during plant DNA extraction is a difficult task because its rigid cell wall is composed of cellulose and other variable content chemical components (such as polysaccharides, polyphenols, and proteins). . The amount of these ingredients varies according to the plant species, plant parts used, environmental conditions and growth stage. Therefore, how to successfully complete the separation of high-quality DNA is inseparable from the credit of these raw materials.

Tris

Tris is a (hydroxymethyl)aminomethane with three primary alcohols and one amine group. It is an effective buffer with a pH between 7 and 9. Together with HCl, it contains a mixture of a weak base and its conjugated weak acid, which can act as a buffer and further increase the permeability of the cell wall. In the process of tissue grinding, when the cell wall and cell membrane rupture, cytoplasmic material is released, so the pH value is changed, thereby destroying the stability of biomolecules (such as nucleic acids). In this case, the buffer plays an important role, and the Tris buffer can maintain the pH of the solution.

EDTA

EDTA chelates divalent cations, such as Mg 2+ and Ca 2+, which are present in enzymes and reduce the enzymatic activity of DNase and RNase. For example, the DNase enzyme requires Mg 2+ ions as a cofactor for its activity. Chelating Mg 2+ ions with EDTA will deactivate the DNase enzyme, thereby protecting DNA. The aggregation of nucleic acids and proteins also requires Mg 2+ ions. Ca 2+ ions are necessary for the consolidation of the middle layer of the cell wall and the stability of the membrane.

Sodium chloride

NaCl helps to remove proteins bound to DNA. By neutralizing the negative charge on the DNA, the molecules can be gathered together, which also helps keep the proteins dissolved in the water layer so that they do not precipitate in alcohol with the DNA.

Osmosis occurs when cells are exposed to hypotonic or hypertonic solutions. If the cells are kept in a hypotonic solution, water will enter the cells, causing swelling, increasing internal pressure and eventually rupturing. On the other hand, in a hypertonic solution, water tends to seep out of the cells, and eventually shrink and shrink the plant cells, leading to cytoplasmic dissolution. Therefore, salt concentration plays an important role in cell lysis.

Ribonuclease A

RNase A is an endoribonuclease that can catalyze the hydrolysis of the 3', 5'-phosphodiester bond of the RNA at the 5'-ester bond in a two-step reaction. The first step is to transfer phosphoric acid to obtain oligonucleotides terminating in pyrimidine 2', 3'-cyclic phosphate. The second is the hydrolysis of cyclic phosphates to produce terminal 3'-phosphates. Because DNA lacks the critical 2'-OH, it cannot be digested by RNase A.

Tris-EDTA (TE) buffer/sterile water

In earlier methods of DNA isolation, DNA used to be stored dry and diluted when needed. Today, for long-term storage, DNA is carefully stored in a buffer that maintains its pH and prevents its degradation. TE buffer contains Tris (10 mM) and EDTA (1 mM), where Tris is the buffer component and EDTA is the chelating component. For DNA separation, the pH is usually set to 7.5-8.5. The weak alkalinity of TE buffer can also prevent the chance of acid hydrolysis, because acid hydrolysis may further damage the stability of DNA in water.

The Tris amino component of TE buffer has the ability to protect DNA strands from radiation damage in solid and fluid solutions. When radiation generates free radicals, it may damage the DNA chain. Therefore, in a fluid solution at ambient temperature, Tris acts by scavenging hydroxyl radicals.

The purpose of EDTA is to chelate Mg 2+ ions in the solution necessary for the action of DNase or RNase, thereby protecting DNA from DNase or RNase.

Sterile water can be used for short-term DNA storage. If TE buffer is used for DNA storage, it should be further diluted with sterile water to dilute the EDTA concentration so that magnesium ions can be used for polymerase activity during PCR, because if the DNA must be sent for sequencing later, then the TE The composition of the buffer will hinder the amplification of DNA.