CN110551711A - Novel technology for extracting mint DNA - Google Patents
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Abstract
the invention discloses a novel technology for extracting mint DNA, which comprises the following steps: cutting fresh herba Menthae plant, and grinding into herba Menthae powder in liquid nitrogen; taking mint powder, adding a mixed solution of diethyl ether and ethanol in an amount which is 2-3 times of the weight of the mint powder, and soaking for 2-3 hours at normal temperature, wherein the weight ratio of diethyl ether: the ratio of ethanol is 1: 1.2-1.5; adding purified water with the same volume into the soak solution in the S2, carrying out hydrothermal treatment at 50-60 ℃ for 1-2h, and centrifuging to obtain a precipitate; taking 5-10g of precipitate, adding 15-20mL of lysis buffer preheated at 65 ℃ and 400 mu L of beta-mercaptoethanol at 300-70 ℃, preserving the temperature for 40-50min at 60-70 ℃, adding 15-20mL of phenol-chloroform-isoamyl alcohol solution with the volume ratio of 25:24:1, mixing, and performing centrifugal separation at room temperature to obtain supernatant; adding isopropanol with the same volume into the supernatant to generate flocculent precipitate, and performing centrifugal separation to obtain DNA precipitate; the DNA precipitate is washed by ethanol with the volume concentration of 75 percent to obtain the mint DNA, thereby achieving the effect of improving the purity of the mint DNA.
Description
Technical Field
The invention relates to the technical field of plant DNA extraction, in particular to a novel technology for extracting mint DNA.
Background
The mint is a labiate plant, about 30 plants in the genus are widely distributed in temperate regions of northern hemisphere. Various plants in Mentha have medicinal value, and can be used in fields of food, medicine, perfume, cosmetics, etc.
the accurate identification of mint varieties is a premise and guarantee of safe application of the mint varieties, the difference and the correlation between the mint varieties are revealed from the DNA molecular level by using molecular markers, more accurate identification can be made on the genetic relationship of the mint varieties, and a basis is provided for variety classification, quality evaluation and the like of mint germplasm resources on the molecular level.
The mint contains a large amount of mint oil, which influences the extraction process of mint DNA, and the mint oil is easy to remain in the mint DNA extract in a large amount, so that the purity of the mint DNA is not high, and further the subsequent use of the mint DNA is influenced.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a novel technology for extracting mint DNA, and the aim of improving the extraction purity of the mint DNA is fulfilled.
The technical purpose of the invention is realized by the following technical scheme:
a novel technique for extracting mint DNA comprises the following steps:
S1 liquid nitrogen grinding: cutting fresh herba Menthae plant, and grinding into herba Menthae powder in liquid nitrogen;
S2 soaking: taking mint powder, adding a mixed solution of diethyl ether and ethanol in an amount which is 2-3 times of the weight of the mint powder, and soaking for 2-3 hours at normal temperature, wherein the weight ratio of diethyl ether: the volume ratio of the ethanol is 1: 1.2-1.5;
S3 hydrothermal treatment: adding purified water with the same volume into the soak solution in the S2, carrying out hydrothermal treatment at 50-60 ℃ for 1-2h, and centrifuging to obtain a precipitate;
S4 DNA extraction: taking 5-10g of precipitate, adding 15-20mL of 65 ℃ preheated lysis buffer and 300-400 mu L of beta-mercaptoethanol, uniformly mixing, and preserving heat at 60-70 ℃ for 40-50min; adding 15-20mL of phenol-chloroform-isoamyl alcohol solution with the volume ratio of 25:24:1, mixing, and performing centrifugal separation at room temperature to obtain supernatant;
s5 DNA isolation: adding isopropanol with the same volume into the supernatant to generate flocculent precipitate, and performing centrifugal separation to obtain DNA precipitate;
S6 DNA wash: washing the DNA precipitate with 75% ethanol by volume to obtain mint DNA.
By adopting the scheme, before DNA extraction, soaking and hydrothermal treatment are carried out in advance, reasonable soaking and hydrothermal treatment parameters are set, so that a large amount of substances such as volatile oil in mint are dissolved in a solution, most mint DNA still exists in a precipitate, the precipitate containing a large amount of mint DNA is obtained through centrifugal separation, and then DNA extraction is carried out, so that the substances such as volatile oil in mint DNA can be greatly reduced, the extraction purity of the mint DNA is improved, the analysis of the mint DNA is facilitated, and bases are provided for variety classification, quality evaluation and the like of mint germplasm resources.
The lysis buffer solution for DNA extraction is 3% CTAB, 100mmol/L Tris-HCl, pH8.0, 20mmol/L EDTA, pH8.0, 1.4mol/L NaCl and 2% beta-mercaptoethanol, Tris-HCl (Ph8.0) provides a buffer environment to prevent nucleic acid from being damaged, EDTA chelates Mg 2+ or Mn 2+ ions to inhibit DNase activity, NaCl provides a high-salt environment to fully dissolve DNA and exists in a liquid phase, and beta-mercaptoethanol is an antioxidant to effectively prevent phenol from being oxidized into quinone and avoid browning.
Preferably, in step S2, the ratio of ethyl ether: the ratio of ethanol is 1: 1.3.
preferably, in step S2, the ultrasonic treatment is assisted, and the ultrasonic power is 50-100W.
by adopting the scheme, through ultrasonic treatment, ultrasonic waves are propagated in water to generate a special 'cavitation effect', countless micro air pockets with the internal pressure reaching thousands of atmospheric pressures are continuously generated, the micro air pockets are continuously 'blasted' to generate microcosmic strong shock waves, and the shock waves continuously act on the mint tissues to further erode the internal structure of the mint tissues, promote escape of substances such as volatile oil and the like, and improve the removal effect of the substances such as the volatile oil and the like.
Preferably, in step S2, the ultrasonic treatment is performed at intervals, each ultrasonic treatment time is 5-8min, and the interval between two adjacent ultrasonic treatments is 20-40 min.
Preferably, in step S2, the ultrasonic power is 80W, each ultrasonic treatment time is 6min, and the interval between two adjacent ultrasonic treatments is 30 min.
By adopting the scheme, the ultrasonic treatment time is too long, so that excessive fragments of mint tissues generated in the ultrasonic treatment process are easily caused, and an escape channel such as volatile oil is blocked and escaped. The ultrasonic treatment time and the interval time are limited, the sufficient flowing time is provided for the mint tissue fragments, the mint tissue fragments are prevented from blocking an escape channel of substances such as the volatile oil, and the removal effect of the volatile oil is further improved. In addition, the ultrasonic wave interval treatment can also reduce energy consumption, and then reduce cost.
preferably, in step S3, the precipitate 8g is taken, 16mL of lysis buffer preheated at 65 ℃ and 320 μ L of beta-mercaptoethanol are added, mixed uniformly, and the temperature is kept at 65 ℃ for 45min; adding 16mL of phenol-chloroform-isoamyl alcohol solution with the volume ratio of 25:24:1, mixing, and centrifuging at room temperature to obtain supernatant.
Object two of the present invention: provides a mint DNA prepared by the method.
In conclusion, the invention has the following beneficial effects:
1. before the extraction of DNA, substances such as a large amount of volatile oil in mint are removed in advance through soaking and hydrothermal treatment, and then the precipitate containing a large amount of mint DNA is subjected to DNA extraction, so that impurities such as volatile oil in mint DNA can be greatly reduced, and the extraction purity of the mint DNA is improved;
2. Ultrasonic auxiliary treatment is carried out in the soaking process, so that escape of the volatile oil and the like is promoted, and the removal effect of the volatile oil and the like is improved;
3. The ultrasonic treatment time and the interval time are limited, the sufficient flowing time is provided for the mint tissue fragments, the mint tissue fragments are prevented from blocking an escape channel of the volatile oil and the like, and the removal effect of the volatile oil and the like is further improved. In addition, the ultrasonic wave interval treatment can also reduce energy consumption, and then reduce cost.
Detailed Description
the present invention will be described in further detail below.
Example 1
A novel technique for extracting mint DNA comprises the following steps:
S1 liquid nitrogen grinding: cutting fresh herba Menthae plant, and grinding into herba Menthae powder in liquid nitrogen;
s2 soaking: taking mint powder, adding a mixed solution of diethyl ether and ethanol in an amount which is 2 times of the weight of the mint powder, and soaking for 2 hours at normal temperature, wherein the weight ratio of diethyl ether: the volume ratio of the ethanol is 1: 1.2; in the period, ultrasonic treatment is assisted, the ultrasonic power is 50W, ultrasonic treatment is carried out twice at intervals, the ultrasonic treatment time is 5min each time, and the interval between two adjacent ultrasonic treatments is 20 min;
s3 hydrothermal treatment: adding purified water with the same volume into the soak solution in the S2, performing hydrothermal treatment for 1h at 50 ℃, and centrifuging to obtain a precipitate;
S4 DNA extraction: taking 5g of precipitate, adding 15mL of 65 ℃ preheated lysis buffer and 300 mu L of beta-mercaptoethanol, uniformly mixing, and preserving heat at 60 ℃ for 40 min; adding 15mL of phenol-chloroform-isoamyl alcohol solution with the volume ratio of 25:24:1, mixing, and performing centrifugal separation at room temperature to obtain supernatant;
s5 DNA isolation: adding isopropanol with the same volume into the supernatant to generate flocculent precipitate, and performing centrifugal separation to obtain DNA precipitate;
s6 DNA wash: washing the DNA precipitate with 75% ethanol to obtain mint DNA,
The proportion of the lysis buffer solution is as follows: 3% CTAB; 100mmol/L Tris-HCl (pH 8.0); 20mmol/L EDTA (pH 8.0); 1.4mol/L NaCl; 2% beta-mercaptoethanol. The preparation process of 100mL lysis buffer is as follows: taking 10% CTAB30mL, adding 10mL of 1mmol/L Tris-HCl (pH8.0), 4mL of 0.5mol/L EDTA (pH8.0), adding 5mol/L NaCl28mL, fixing the volume to 100mL, autoclaving for 20min, and adding beta-mercaptoethanol at present.
example 2
S1 liquid nitrogen grinding: cutting fresh herba Menthae plant, and grinding into herba Menthae powder in liquid nitrogen;
s2 soaking: taking mint powder, adding a mixed solution of diethyl ether and ethanol in an amount which is 2.5 times the weight of the mint powder, and soaking the mint powder at normal temperature for 2.5 times, wherein the weight ratio of diethyl ether: the volume ratio of the ethanol is 1: 1.3; in the period, ultrasonic treatment is assisted, the ultrasonic power is 80W, ultrasonic treatment is carried out twice at intervals, the ultrasonic treatment time is 6min each time, and the interval between two adjacent ultrasonic treatments is 30 min;
S3 hydrothermal treatment: adding purified water with the same volume into the soak solution in the S2, performing hydrothermal treatment for 1.5h at 55 ℃, and centrifuging to obtain a precipitate;
S4 DNA extraction: taking 8g of precipitate, adding 16mL of lysis buffer preheated at 65 ℃ and 320 mu L of beta-mercaptoethanol, uniformly mixing, and preserving heat at 65 ℃ for 45min; adding 16mL of phenol-chloroform-isoamyl alcohol solution with the volume ratio of 25:24:1, mixing, and performing centrifugal separation at room temperature to obtain supernatant;
S5 DNA isolation: adding isopropanol with the same volume into the supernatant to generate flocculent precipitate, and performing centrifugal separation to obtain DNA precipitate;
S6 DNA wash: washing the DNA precipitate with 75% ethanol to obtain mint DNA,
the formulation and configuration of the lysis buffer was the same as in example 1.
Example 3
S1 liquid nitrogen grinding: cutting fresh herba Menthae plant, and grinding into herba Menthae powder in liquid nitrogen;
S2 soaking: taking mint powder, adding a mixed solution of diethyl ether and ethanol in an amount which is 3 times of the weight of the mint powder, and soaking for 3 hours at normal temperature, wherein the weight ratio of diethyl ether: the volume ratio of the ethanol is 1: 1.5; in the period, ultrasonic treatment is assisted, the ultrasonic power is 100W, ultrasonic treatment is carried out twice at intervals, the ultrasonic treatment time is 8min each time, and the interval between every two adjacent ultrasonic treatments is 40 min;
s3 hydrothermal treatment: adding purified water with the same volume into the soak solution in the S2, performing hydrothermal treatment for 2h at 60 ℃, and centrifuging to obtain a precipitate;
s4 DNA extraction: taking 10g of precipitate, adding 20mL of 65 ℃ preheated lysis buffer and 400 mu L of beta-mercaptoethanol, uniformly mixing, and keeping the temperature at 70 ℃ for 50min; adding 20mL of phenol-chloroform-isoamyl alcohol solution with the volume ratio of 25:24:1, mixing, and performing centrifugal separation at room temperature to obtain supernatant;
s5 DNA isolation: adding isopropanol with the same volume into the supernatant to generate flocculent precipitate, and performing centrifugal separation to obtain DNA precipitate;
s6 DNA wash: washing the DNA precipitate with 75% ethanol to obtain mint DNA,
the formulation and configuration of the lysis buffer was the same as in example 1.
example 4 differs from example 2 in that: in S2, ultrasonic processing is not assisted.
example 5 differs from example 2 in that: the sonication in S2 was not performed at intervals, but continued for 12 min.
Example 6 differs from example 2 in that: ether in S2: the ratio of ethanol is 1: 1.2.
Example 7 differs from example 2 in that: ether in S2: the ratio of ethanol is 1: 1.5.
Determination of mint DNA concentration and purity: by adopting a conventional method, using a UV-1810 type ultraviolet-visible spectrophotometer to respectively determine the light absorption values OD260 and OD280 of the mint DNA sample at 260nm and 280nm, wherein the DNA purity calculation formula is as follows:
The assay data are listed in table 1.
table 1 DNA purity measurement data.
The OD260/OD280 of the pure DNA is approximately equal to 1.8, and the closer the OD260/OD280 value is to 1.8, the higher the DNA purity is. As can be seen from the test results in Table 1, the OD260/OD280 values of examples 1-3 are closer to 1.8, which shows that the purity of the mint DNA prepared by the method of the present invention is higher, and the soaking and ultrasonic treatment effectively improves the purity of the mint DNA. As can be seen from comparison of examples 2 and 5, the extraction purity of the mint DNA was also improved by the ultrasonic wave interval treatment. As can be seen from comparative examples 6 and 7, the ratio of diethyl ether: the ratio of ethanol influences the extraction purity of the mint DNA, since the appropriate ether: the ratio of ethanol can improve escape and dissolution of volatile oil, and further improve extraction purity of herba Menthae DNA.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.
Claims (7)
1. a novel technology for extracting mint DNA is characterized in that: the method comprises the following steps:
s1 liquid nitrogen grinding: cutting fresh herba Menthae plant, and grinding into herba Menthae powder in liquid nitrogen;
S2 soaking: taking mint powder, adding a mixed solution of diethyl ether and ethanol in an amount which is 2-3 times of the weight of the mint powder, and soaking for 2-3 hours at normal temperature, wherein the weight ratio of diethyl ether: the volume ratio of the ethanol is 1: 1.2-1.5;
s3 hydrothermal treatment: adding purified water with the same volume into the soak solution in the S2, carrying out hydrothermal treatment at 50-60 ℃ for 1-2h, and centrifuging to obtain a precipitate;
S4 DNA extraction: taking 5-10g of precipitate, adding 15-20mL of lysis buffer preheated at 65 ℃ and 400 mu L of beta-mercaptoethanol at 300-70 ℃, uniformly mixing, preserving the heat at 60-70 ℃ for 40-50min, adding 15-20mL of phenol-chloroform-isoamylol solution with the volume ratio of 25:24:1, mixing, and performing centrifugal separation at room temperature to obtain supernatant;
s5 DNA isolation: adding isopropanol with the same volume into the supernatant to generate flocculent precipitate, and performing centrifugal separation to obtain DNA precipitate;
S6 DNA wash: washing the DNA precipitate with 75% ethanol by volume to obtain mint DNA.
2. The novel process for extracting mint DNA according to claim 1, wherein: in step S2, diethyl ether: the volume ratio of ethanol is 1: 1.3.
3. the novel process for extracting mint DNA according to claim 1, wherein: in step S2, ultrasonic treatment is assisted, and the ultrasonic power is 50-100W.
4. The novel process for extracting mint DNA according to claim 3, wherein: in step S2, ultrasonic wave interval treatment is carried out for multiple times, each ultrasonic wave treatment time is 5-8min, and the interval between two adjacent ultrasonic wave treatments is 20-40 min.
5. the novel process for extracting mint DNA according to claim 4, wherein: in step S2, the ultrasonic power is 80W, the ultrasonic treatment time is 6min each time, and the interval between two adjacent ultrasonic treatments is 30 min.
6. the novel process for extracting mint DNA according to claim 1, wherein: in step S3, 8g of precipitate is taken, 16mL of lysis buffer preheated at 65 ℃ and 320 μ L of beta-mercaptoethanol are added, the mixture is uniformly mixed, the temperature is kept at 65 ℃ for 45min, 16mL of phenol-chloroform-isoamyl alcohol solution with the volume ratio of 25:24:1 is added, and after mixing, the supernatant is obtained by centrifugal separation at room temperature.
7. a mint DNA prepared by the method of any one of claims 1 to 6.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1742093A (en) * | 2002-11-18 | 2006-03-01 | 新加坡科技研究局 | Method and system for cell and/or nucleic acid molecules isolation |
| CN101100479A (en) * | 2007-08-03 | 2008-01-09 | 安徽大学 | Method for extracting residual DNA in edible vegetable oil |
| CN102433325A (en) * | 2012-01-17 | 2012-05-02 | 扬州大学 | Extraction method of RNA (ribonucleic acid) of rape seeds |
| CN106047860A (en) * | 2016-06-03 | 2016-10-26 | 中国农业科学院特产研究所 | Plant genome DNA extracting method |
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- 2018-05-31 CN CN201810555818.8A patent/CN110551711A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1742093A (en) * | 2002-11-18 | 2006-03-01 | 新加坡科技研究局 | Method and system for cell and/or nucleic acid molecules isolation |
| CN101100479A (en) * | 2007-08-03 | 2008-01-09 | 安徽大学 | Method for extracting residual DNA in edible vegetable oil |
| CN102433325A (en) * | 2012-01-17 | 2012-05-02 | 扬州大学 | Extraction method of RNA (ribonucleic acid) of rape seeds |
| CN106047860A (en) * | 2016-06-03 | 2016-10-26 | 中国农业科学院特产研究所 | Plant genome DNA extracting method |
Non-Patent Citations (4)
| Title |
|---|
| 周东坡等: "《生物制品学》", 31 October 2006, 化学工业出版社 * |
| 徐锐等: "《发酵技术》", 31 January 2016, 重庆大学出版社 * |
| 曹墨菊等: "《植物生物学技术概论》", 30 September 2017, 中国农业大学出版社 * |
| 衷友泉等: "《中医药基础化学实验》", 31 July 2017, 中国协和医科大学出版社 * |
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Application publication date: 20191210 |