Background and overview[1]
Phenol-chloroform-isoamyl alcohol mixture is often used as analytical extraction solution for extracting DNA, polysaccharides, etc.
Preparation[1-4]
Application examples of phenol-chloroform-isoamyl alcohol mixtures are as follows:
1) For the extraction of genomic DNA of sugar-producing bacteria suitable for whole-genome sequencing, add extraction buffer and lysozyme to the culture of sugar-producing bacteria, let stand at 37°C for 10 minutes, and then add Proteinase K, let stand at 65°C for 1 hour; add a mixed solution of phenol-chloroform-isoamyl alcohol and extract; add a mixed solution of chloroform-isoamyl alcohol and extract; add cold ethanol and sodium acetate, centrifuge, and wash the precipitate with ethanol Then dry at room temperature, add DNA extraction buffer to dissolve; add RNase, and let stand at 37°C for 10 minutes; add PEG6000, mix, and let stand at 50°C for 10 minutes; add a mixed solution of phenol-chloroform-isoamyl alcohol, and extract; Add chloroform-isoamyl alcohol mixed solution and extract; add cold ethanol and sodium acetate solution, centrifuge, wash the precipitate with ethanol and dry at room temperature, add ddH2O or TE buffer to dissolve, which is the extracted DNA solution.
2) A method for extracting high-quality genomic DNA from Ilex polygonum, including the following steps: (1) Quick-freeze the plant material in liquid nitrogen and grind it into powder; (2) Add DNA extraction solution and add proteinase K to 20 μg/mL, Incubate at 65°C; (3) Centrifuge to collect the supernatant, add an equal volume of phenol: chloroform: isoamyl alcohol mixture to extract, then centrifuge to collect the supernatant; (4) Add 0.6 to 0.8 times the volume of isopropyl alcohol to precipitate DNA ; Wash with ethanol, partially dry and dissolve in TE buffer to obtain a crude DNA extract; (5) Add NaCl to the crude DNA extract to 0.5 mol/L, then add CTAB/NaCl solution, and add RNase-A until final The concentration is 10 μg/mL, incubated at 65°C; (6) Extract with equal volumes of phenol: chloroform: isoamyl alcohol mixture and equal volume of chloroform: isoamyl alcohol mixture, and collect the supernatant; (7) Add 0.6~0.8 Double the volume of isopropyl alcohol to precipitate DNA, then wash with ethanol, partially dry and dissolve in TE buffer to obtain a high-quality DNA extraction solution. The present invention can extract high-quality genomic DNA from Ambrosia elata.
3) A method for extracting and purifying total bacteria from environmental samples, which includes the following steps: take the environmental sample, add water or buffer, and mix; add phenol-chloroform-isoamyl alcohol mixture, and mix , wait until the solvent is separated, remove the upper layer and set aside; centrifuge the removed upper layer, discard the supernatant, and keep the precipitate; wash the precipitate until it is almost colorless, and resuspend the bacterial solution. The method of the present invention simplifies the pre-processing steps of PCR detection, eliminates the cumbersome DNA extraction steps, and eliminates interfering substances in the PCR reaction. The diluted bacterial liquid or colony obtained by the method is directly used as a template for PCR amplification, which is highly sensitive and , good specificity, short detection time, greatly reducing detection costs.
4) An extraction method of purslane polysaccharide. The method includes the following steps: (1) After the raw purslane medicinal material is crushed, use ethanol or petroleum ether to carry out reflux extraction twice, each time for 2 hours, suction filtration, and drying to obtain purslane medicinal powder for later use. (2) Purslane medicinal powder is placed in an autoclave for extraction. The temperature range is set to 105-121°C and the extraction time is 1 hour. (3) Extract the extracted crude polysaccharide solution with equal volumes of phenol:chloroform:isoamyl alcohol (25:24:1) to remove the protein, retain the supernatant aqueous solution, rotary evaporate the solution, and use absolute ethanol Precipitate overnight and centrifuge to obtain polysaccharides. The yield of polysaccharides extracted by the method of the present invention is 89%-96% higher than that extracted by the traditional water bath method. At the same time, the equipment used in the method of the present invention is simple and easy to obtain, the method of purifying polysaccharides is simple and reproducible, and the entire extraction cost is low. , suitable for large-scale industrial production.
Main reference materials
[1]CN201210145537.8 A method for extracting genomic DNA of sugar-producing bacteria suitable for whole-genome sequencing
[2] CN201510875261.2 A method of extracting genomic DNA from Ammodium elata
[3]CN201611152634.4 A method for extracting and purifying total bacteria from environmental samples
[4]CN201810700186.X An extraction method of purslane polysaccharide
