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Improved CTAB method for extracting polygonum capitatum DNA

A technology for extracting supernatant from Polygonum cephalum, applied in the field of DNA extraction, can solve the problems of high cost, complicated extraction process, and low purity, and achieve the effect of short extraction cycle, simple AB method, and high extraction purity

Inactive Publication Date: 2015-10-21
AFFILIATED YONGCHUAN HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the traditional DNA extraction method of Polygonum cephalum still has the following problems: the extraction process is complicated, the cost is high, the cycle is long, the purity is low, and the primers are not standardized.

Method used

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  • Improved CTAB method for extracting polygonum capitatum DNA
  • Improved CTAB method for extracting polygonum capitatum DNA
  • Improved CTAB method for extracting polygonum capitatum DNA

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Embodiment

[0042] Take the young leaves of Polygonum cephalosa from different sources (see Table 1), and store them in a low-temperature refrigerator at -70°C for later use. Take 2 g of the frozen Polygonum cephalosa material, put it into a frozen mortar immediately, and quickly spread it evenly on the leaves. Sprinkle 40-50mg of DIECA crystals, add liquid nitrogen, and quickly grind into powder; quickly transfer the powder into a 1.5ml centrifuge tube, add 600μl 65°C preheated CTAB extraction buffer, shake well, 65°C water bath for 60min, Mix 10-15 times during this period. Take the supernatant, add an equal volume of chloroform:isoamyl alcohol (24:1, V / V), invert and mix 10-15 times, transfer the supernatant to another 1.5ml centrifuge tube, and take the supernatant again Extract with chloroform: isoamyl alcohol (24:1, V / V) once. Weigh 24.608g of anhydrous sodium acetate, add 70ml of double-distilled water and heat to 50-80°C to dissolve, then add double-distilled water to make up to ...

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Abstract

The invention relates to an improved CTAB method for extracting polygonum capitatum DNA. The method comprises the following steps: fetching polygonum capitatum, grinding polygonum capitatum into powder in liquid nitrogen, transferring the powder to a centrifuge tube, adding a CTAB extracting buffering solution, fully shaking the centrifuge tube, carrying out water bath and centrifugation; obtaining the supernate, adding chloroform and isoamyl alcohol with an equal volume, transferring the supernate to another centrifuge tube, collecting the supernate again, extracting the supernate by chloroform and isoamyl alcohol; adding NaAc and isopropanol into supernate, slightly turning over the centrifuge upside down to evenly mix the solution, carrying out centrifugation, discarding the supernate, washing the precipitate by ethanol (70%), carrying out centrifugation, absorbing ethanol by a small gun head, drying the precipitate in the air; removing the ethanol in precipitate, dissolving and digesting the precipitate by TE containing RNA enzymes until the RNA is completely degraded; extracting the solution by phenol, chloroform, and isoamyl alcohol with an equal volume respectively, adding supernate into NaAc and cold anhydrous ethanol, slightly and evenly mixing, carrying out centrifugation, washing the precipitate by an ethanol solution (70%), drying the precipitate in the air, adding a TD solution to dissolve the precipitate, and finally preserving the solution at a low temperature. The modified CTAB method is used to extract polygonum capitatum DNA and has the advantages of simpleness, low cost, and higher purity of polygonum capitatum DNA.

Description

technical field [0001] The invention relates to a DNA extraction method, in particular to an improved CTAB method for extracting Polygonum cephalum DNA. Background technique [0002] At present, the research on Polygonum cephalum is mainly in its chemical composition and pharmacological action, clinical application, microscopic identification and cultivation techniques. Different research groups compared the total flavonoid content of wild and cultivated Polygonum cephalum in different origins and found that there are some differences, suggesting that the growth environment has an impact on the quality of medicinal materials. In recent years, with the increase in market value of Polygonum cephalum, the research on the molecular biology of Polygonum cephalum has gradually entered a hot stage. With people's attention to the quality of medicines, in order to ensure the quality of traditional Chinese medicines, the use of modern molecular biology DNA barcode technology to ident...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 何翠马海东
Owner AFFILIATED YONGCHUAN HOSPITAL OF CHONGQING MEDICAL UNIV
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