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Extraction of DNA in Pleurotus eryngii by improved CTAB (cetyltrimethylammonium bromide) method

A technology of Pleurotus eryngii and extraction method, which is applied in the direction of DNA preparation, recombinant DNA technology, etc., and can solve problems such as polysaccharide pollution

Active Publication Date: 2011-12-21
湖南果秀食品有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to effectively solve the problem of polysaccharide contamination in DNA separation and purification

Method used

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  • Extraction of DNA in Pleurotus eryngii by improved CTAB (cetyltrimethylammonium bromide) method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] (1) Take 0.5g of Pleurotus eryngii mycelium ball, add liquid nitrogen (equivalent to 1 / 2 of the volume of the mortar) and grind it into a fine powder, put it in a 10ml centrifuge tube, add 3% CTAB extraction buffer preheated at 65°C 4ml of solution (100mmol / LTris-HCl(pH8.0), 20mmol / LEDTA(pH8.0), 1.4mol / LNaCl, 3%CTAB, 2%PVP, 2.67%β-mercaptoethanol), shake and mix, put Incubate in a water bath at a constant temperature of 65°C for 1 hour, oscillate and mix once every 10-15 minutes; take out and centrifuge for 10 minutes; take the supernatant after centrifugation;

[0016] (2) Add absolute ethanol equivalent to 30% of the volume of CTAB added for the first time to the supernatant; then add 4 mL of a mixed solution of Tris-phenol, chloroform, and isoamyl alcohol, and the Tris-phenol:chloroform in the mixed solution : isoamyl alcohol=25:24:1, centrifuged for 10min; discard the precipitate and get the supernatant;

[0017] (3) Add 4 mL of a mixed solution of chloroform and i...

Embodiment 2

[0024] Take 0.5g of Pleurotus eryngii hyphae ball, add liquid nitrogen (equivalent to 1 / 2 of the volume of the mortar) and grind it into a fine powder, put it in a 10ml centrifuge tube, add 4ml of 3% CTAB extraction buffer preheated at 65°C ( 100 mmol / LTris-HCl(pH8.0), 20mmol / LEDTA(pH8.0), 1.4mol / LNaCl, 3%CTAB, 2%PVP, 2.67%β-mercaptoethanol), shake and mix well, put in constant temperature 65 Keep warm in a water bath at ℃ for 1 hour, shake and mix once every 10-15 minutes; take out and centrifuge for 10 minutes; take the supernatant after centrifugation;

[0025] In the supernatant, add the dehydrated alcohol that is equivalent to 25% volume of CTAB amount added for the first time; Alcohol=26:23:2, centrifuge for 10min; discard the precipitate and take the supernatant;

[0026] In the supernatant, add the mixed solution of 4mL chloroform and isoamyl alcohol to extract again, in the mixed solution, chloroform:isoamyl alcohol=28:3; centrifuge for 10min, discard the precipitate...

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Abstract

The invention relates to extraction of DNA in Pleurotus eryngii by an improved CTAB (cetyltrimethylammonium bromide) method. 25-35% anhydrous alcohol is added while adding a mixed solution of Tris-phenol, chloroform and isoamyl alcohol, and the OD260 / OD280 values of the extracted DNA are respectively 1.9-2.0, which indicates that the addition of the alcohol is beneficial to removing proteins. Since the alcohol is dissolvable in water and has great affinity for water, the hydration shells of the protein granules can be destroyed, so the proteins precipitate at the isoelectric point; and therefore, the protein precipitate can be removed in the extraction process. When the DNA is subjected to ultraviolet detection by five different methods, the result indicates that only the DNA tape extracted by the invention is the brightest, basically has no degradation trailing phenomenon, and only has few substances detained near the sample charging hole, which indicates that the low-concentration alcohol precipitation method can well remove polysaccharides in the DNA.

Description

technical field [0001] The invention relates to a method for extracting Pleurotus eryngii DNA. Especially a CTAB method to extract the DNA of Pleurotus eryngii. Background technique [0002] At present, DNA extraction, separation and purification are one of the important technologies in the genetic engineering research of edible fungi. However, edible and medicinal fungi are often rich in polysaccharides, and many of the physical and chemical properties of polysaccharides are similar to nucleic acids, so how to remove the polysaccharides in the extraction method of edible fungi DNA is a thorny problem; because polysaccharides can inhibit the restriction of DNA Therefore, DNA samples contaminated with polysaccharides cannot be used for further molecular biology research. Existing CTAB method extracts the DNA in the plant gene tissue, all is to add PVP in the CTAB buffer solution to remove a little polysaccharide; + or K + Polysaccharides were removed by Tris-phenol, chlor...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 阳国秀易恢满夏志兰姬建军
Owner 湖南果秀食品有限公司
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