CN114426967A - Non-toxic plant universal high-purity genome DNA rapid extraction method and kit - Google Patents
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Abstract
The invention discloses a non-toxic plant universal high-purity genome DNA rapid extraction method, which comprises the following steps: (1) grinding and crushing a plant sample to be extracted; (2) adding FN-PW cell rinsing solution to rinse and remove impurities such as polysaccharide, polyphenol and pigment; (3) adding FN-S cell lysate to release genome DNA, centrifuging and taking supernatant; (4) adding isopropanol to resuspend the precipitate, centrifuging and discarding the supernatant; (5) adding 70-75% ethanol for heavy suspension precipitation, centrifuging and discarding the supernatant; (6) adding absolute ethyl alcohol to resuspend and precipitate, centrifuging and discarding supernatant; (7) and after drying, adding sterile ultrapure water or TE for resuspension and precipitation, centrifuging and taking supernatant to obtain the high-purity genome DNA solution. The method has the advantages of low extraction cost, simple operation, capability of quickly and safely extracting the genome DNA from the multiple plants, good integrity, high yield and high purity of the extracted genome DNA, and capability of being applied to various downstream molecular biological experiments such as gene cloning, molecular marking, enzyme digestion, library construction, Real-time PCR, Southern Blot and the like.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a non-toxic plant universal high-purity genome DNA rapid extraction method and a kit.
Background
The extraction of plant genome DNA is the basis of plant molecular biology research, high-quality genome DNA can be directly applied to various downstream molecular biology experiments such as gene cloning, molecular marking, enzyme digestion, library construction, Real-time PCR and Southern Blot, however, the existing plant genome DNA extraction technologies at home and abroad at least have a plurality of defects shown in Table 1:
TABLE 1 deficiencies and specific manifestations of various existing plant genomic DNA extraction techniques at home and abroad
Therefore, it is an urgent need to develop a method for extracting high-purity genomic DNA from plants that can overcome the above-mentioned deficiencies of the prior art.
Disclosure of Invention
In view of the above, the present invention aims to provide a non-toxic plant-universal method and kit for rapidly extracting high-purity genomic DNA, so as to overcome the problems in the prior art. The method provided by the invention has the advantages of low extraction cost and simple operation, does not use toxic reagents and expensive reagents, consumables or instrument equipment in the extraction process, can quickly, efficiently and safely extract the genome DNA from multiple plants, has good integrity, high yield and high purity of the extracted genome DNA, and can be directly applied to various downstream molecular biology experiments such as gene cloning, molecular marking, enzyme digestion, library construction, Real-time PCR, Southern Blot and the like.
The invention provides a non-toxic plant universal high-purity genome DNA rapid extraction method, which comprises the following steps:
(1) grinding and crushing a plant sample to be extracted;
(2) adding FN-PW cell rinsing liquid to rinse and remove impurities such as polysaccharide polyphenol, pigment and the like in a sample, wherein the FN-PW cell rinsing liquid consists of 0.5-4% (w/v) PVP, 0-2% (w/v) L-cysteine, 0.5-4% (v/v) Triton X-100 and pure water;
(3) adding FN-S cell lysate to release genomic DNA, and centrifugingCollecting supernatant, wherein the FN-S cell lysate is prepared from 1-10% (w/v) CTAB, 10-300 mM Tris-HCl (pH 7.5-8.5), and 1-50 mM Na2EDTA (the pH value is 7.5-8.5), 1.4-2.0M NaCl, 0-2% (w/v) PVP, 0.5-2% (v/v) Triton X-100 and pure water;
(4) adding isopropanol into the supernatant obtained in the step (3) for heavy suspension precipitation, oscillating, centrifuging and removing the supernatant;
(5) adding 70-75% ethanol for heavy suspension precipitation, oscillating, centrifuging and discarding supernatant;
(6) adding absolute ethyl alcohol to resuspend and precipitate, oscillating, centrifuging and removing supernate;
(7) drying the centrifugal precipitate obtained in the step (6), adding sterile ultrapure water or TE for heavy suspension precipitation, centrifuging and taking supernatant fluid to obtain a high-purity genome DNA solution;
wherein, the step (2) is an optional step, and the step can be omitted for samples with less impurities, such as rice fresh leaves, corn fresh leaves, wheat fresh leaves, various plant root tips and the like; for samples rich in impurities such as polysaccharide polyphenol, pigment and the like, such as cotton mature leaves, rice dry-cured leaves, citrus leaves, apple tree leaves, poplar leaves and the like, the step cannot be omitted, and the rinsing times are properly increased according to the actual conditions of the samples, for example, the rinsing times of 100mg of cotton mature leaves are more than 5 times, so that the polysaccharide polyphenol and the pigment can be effectively removed; in the step (3), whether RNase A is added or not can be selected according to the purpose of an experiment when the genomic DNA is released, and the RNase A does not need to be added when the obtained genomic DNA is only used for a short fragment PCR amplification template below 2kb, for example, the RNase A does not need to be added when the arabidopsis genomic DNA is extracted for T-DNA insertion identification; the obtained genomic DNA is used for cloning short fragment genes of 2kb or less, and is added with RNase A when used for downstream molecular biology experiments such as enzyme digestion, library construction, Real-time PCR and Southern Blot.
In the invention, the amount of the sample is not fixed and can be taken according to actual requirements, when the amount of the sample is less than 300mg, the experiment can be completed by using a 2ml centrifugal tube, and when the amount of the sample exceeds 300mg, the 2ml centrifugal tube can be replaced by a round-bottom centrifugal tube with a larger range for experiment.
In the invention, in the step (1), when the plant sample to be extracted is ground and crushed, different grinding modes can be selected according to the characteristics of the sample and the experimental conditions for grinding and crushing, if the plant sample to be extracted can be ground into powder and then sampled to a centrifugal tube for subsequent operation, the plant sample to be extracted can also be directly added into the centrifugal tube for sample grinding and crushing and then subsequent operation is carried out.
In the present invention, in the step (2), when the FN-PW cell rinsing solution is added to rinse and remove impurities such as polysaccharide polyphenol and pigment in the sample, the rinsing method can be appropriately adjusted according to the condition of the impurities such as polysaccharide polyphenol and pigment contained in the plant sample to be extracted, for example, the amount of the FN-PW cell rinsing solution, the rinsing method and the rinsing temperature can be appropriately adjusted, and preferably, the following method can be adopted for rinsing: adding FN-PW cell rinsing solution preheated to 50-75 ℃ into the crushed plant sample to be extracted according to the dosage of 0.02ml/mg (based on the weight of the plant sample to be extracted), shaking and rinsing up and down for 30 seconds, centrifuging at 12000-14000 rpm for 1min, and discarding the supernatant.
In the present invention, in the step (3), when the FN-S cell lysate is added to release the genomic DNA, the experimental parameters, such as the amount of FN-S cell lysate used, the experimental temperature, the experimental time, etc., can be appropriately adjusted according to the experimental requirements and experimental results, and preferably, when the obtained genomic DNA is used only for PCR amplification templates with short fragments of 2kb or less, the genomic DNA can be released in the following manner: adding FN-S cell lysate which is preheated to 50-75 ℃ according to the dosage of 4ul/mg (based on the weight of a plant sample to be extracted) for heavy suspension precipitation, oscillating up and down for 5S, and bathing for 2-5 min at 50-75 ℃; when the obtained genomic DNA is used for downstream molecular biology experiments such as enzyme digestion, library construction, Real-time PCR and Southern Blot besides the cloning of the following short-fragment genes of 2kb, the genomic DNA can be released in the following way: adding FN-S cell lysate which is preheated at 50-75 ℃ according to the dosage of 4ul/mg (based on the weight of a plant sample to be extracted) for resuspending precipitation, adding RNase A according to the dosage of 1:0.02(v/v, based on the dosage of the FN-S cell lysate), oscillating the mixture up and down for 5S, and carrying out warm bath at 50-75 ℃ for 5-15 min.
In the invention, in the step (4), the isopropanol is added mainly for removing polysaccharide and CTAB impurities while precipitating nucleic acid in a high-salt solution, the dosage of the isopropanol can be selected according to the sample amount of the plant sample to be extracted and the amount of impurities contained in the plant sample, and preferably, the addition amount of the isopropanol is 0.6-1 time of the volume of the supernatant obtained in the step (3).
In the invention, in the step (5), the 70% -75% ethanol is added for resuspension and precipitation, which is mainly used for precipitating nucleic acids and removing impurities, the amount of the 70% -75% ethanol can be selected according to the amount of the plant sample to be extracted and the amount of the impurities contained in the plant sample, and the number of times of adding the 70% -75% ethanol for resuspension and precipitation can be selected, preferably, the 70% -75% ethanol is added for resuspension and precipitation according to the amount of 8-16 ul/mg (based on the weight of the plant sample to be extracted).
In the invention, in the step (6), the addition of the absolute ethyl alcohol for resuspension and precipitation is mainly to precipitate nucleic acids and remove impurities, the amount of the absolute ethyl alcohol can be selected and the number of times of adding the absolute ethyl alcohol for resuspension and precipitation can be selected according to the sample amount of the plant sample to be extracted and the amount of the impurities contained in the plant sample, and preferably, the absolute ethyl alcohol is added according to the amount of 8-16 ul/mg (based on the weight of the plant sample to be extracted) for resuspension and precipitation.
In the present invention, in the step (7), when the centrifugal precipitate obtained in the step (6) is dried, an appropriate drying method can be determined according to experimental conditions, and preferably, the drying can be performed as follows: and opening the cover of the centrifuge tube with the precipitate, and placing the centrifuge tube in a forced air drying oven at 65-75 ℃ for airing for 1-5 min.
In the invention, when sterile ultrapure water or TE heavy suspension precipitation is added in the step (7), preheated sterile ultrapure water or TE heavy suspension precipitation is preferably selected, so that a DNA solution can be quickly obtained, impurities are centrifugally precipitated at the bottom of a tube, and a supernatant (DNA solution) is taken for storage, thereby effectively preventing the DNA solution from browning or breeding mold.
The invention also provides a non-toxic plant general-purpose high-purity genomic DNA rapid extraction kit, wherein the reagent contains the FN-PW cell rinsing liquid or the FN-S cell lysis liquid or contains the FN-PW cell rinsing liquid and the FN-S cell lysis liquid simultaneously.
The invention has the beneficial effects that:
(1) the universality is strong: the genomic DNA extraction method provided by the invention can be universally used in most plant samples, including but not limited to fresh plant samples, dried plant samples and plant specimens; the method for extracting the genome DNA provided by the invention can be universally used in samples rich in impurities such as polysaccharide polyphenol, grease or pigment, and the like, including but not limited to cotton mature leaves, potato tubers and woody plant leaves, and is characterized in that the FN-PW cell rinsing liquid can effectively remove the impurities in the samples from the source before the genomic DNA is released.
(2) The application range is wide: the genomic DNA extracted by the method can be applied to various downstream molecular biology experiments such as gene cloning, molecular marking, enzyme digestion, library construction, Real-time PCR, Southern Blot and the like.
(3) The extraction speed is high: the method for extracting the genome DNA can finish the work of extracting the genome DNA of a single sample within 10-30 min, and is characterized in that a proper amount of Triton X-100 is contained in FN-S cell lysate, the combination of CTAB (cetyl trimethyl ammonium bromide) with protein and polysaccharide is accelerated under the condition of 50-75 ℃ warm bath, the DNA is rapidly released, and the agitation and the uniform mixing are not needed in the warm bath process, so the extraction speed is obviously improved, while the traditional CTAB method needs to be subjected to at least warm bath for 30min under the condition of 65 ℃, and needs to be oscillated for several times every few minutes in the period.
(4) The extraction purity is high: the method of the invention extracts the genomic DNA of different plant samples, the OD thereof260/OD280The average value of (A) is 1.8 to 2.0; the genome DNA extracted by the method can be seen as a clear DNA band by 1 percent agarose gel electrophoresis.
(5) The extraction yield is high: the yield of the high-purity genome DNA extracted from a fresh plant sample of 100mg by the method is more than 15 ug.
(6) The extraction steps have high flexibility: the method is applied to the sample containing less impurities, and the FN-PW cell rinsing liquid is not required to be used but is not limited to be used, when the method is applied to the sample containing more impurities, the rinsing times of the FN-PW cell rinsing liquid can be flexibly selected according to the quantity of the sample and the characteristics of the sample; the method can extract the genome DNA from a trace sample, a small amount of sample or a large amount of sample, and only needs to add FN-S cell lysate according to the sample amount of 1:4 (w/v); the method can select whether to add the RNase A and different warm bath time according to the experimental purpose, when the method is applied to the preparation of the short fragment PCR amplification template with the length of less than 2kb, the RNase A does not need to be added, and the warm bath lasts for 2-5 min, and when the method is applied to downstream molecular biology experiments such as enzyme digestion, library construction, Real-time PCR and Southern Blot which have higher requirements on the quality of genome DNA, the RNase A needs to be added, and the warm bath lasts for 5-15 min.
(7) The extracted genome DNA is not easy to deteriorate and degrade after being stored for a long time: the method of the invention uses preheated sterile ultrapure water or TE for heavy suspension precipitation, can quickly obtain DNA solution with insoluble impurities, further centrifugally precipitate the impurities at the bottom of the tube, and take supernatant (DNA solution) for storage, can effectively prevent the DNA solution from browning or breeding mould caused by the impurity precipitates, prevent DNA degradation, and overcome the defects of low DNA dissolution speed and large amount of impurity precipitates in the DNA solution of the traditional extraction method.
(8) The dosage of extraction reagent is less: the method extracts high-purity genome DNA from 50mg of fresh plant samples containing less impurities, only needs 200ul of FN-S cell lysate, 4ul of RNase A, 200ul of isopropanol, 2ml of absolute ethyl alcohol and a very small amount of sterile ultrapure water or TE, and is beneficial to popularization and application.
(9) The extraction cost is low: according to the method, the 50mg fresh plant sample is extracted without using relatively expensive consumables such as magnetic beads, adsorption columns, centrifugal columns, sampling cards and the like, only 12 ml centrifugal tube and 2 1.5ml centrifugal tubes are needed, and the consumable cost is about 1/50 of that of a centrifugal column method; the method does not need expensive chemical reagents and has little dosage of extraction reagents; the method does not need expensive instruments and equipment, and the used instruments and equipment are conventional instruments and equipment in a molecular biology laboratory; although the method does not use the expensive instruments, reagents and consumables, the extraction effect of the method can be achieved by using magnetic beads, adsorption columns, centrifugal columns, sampling cards and other adsorption or filtration means, and the method can also be applied to high-throughput genome DNA automatic extraction equipment and belongs to the protection range of the method.
(10) The extraction process is safe and nontoxic: the method does not use strong acid or strong alkali and other strong corrosive reagents, does not use chloroform, phenol, mercaptoethanol, guanidine isothiocyanate or DTT and other toxic reagents, and overcomes the defect that the traditional genomic DNA extraction method uses toxic reagents or strong corrosive reagents.
Drawings
FIG. 1 is a 1% agarose gel electrophoresis of genomic DNA of rice leaves extracted by the method of the present invention, wherein lane 1 is Marker (BM2000 DNA Marker), and lanes 2-11 are genomic DNA extracted from different rice plants;
FIG. 2 is a 1% agarose gel electrophoresis of tomato leaf genome DNA extracted by the method of the present invention, wherein lane 1 is Marker (BM5000 DNA Marker), and lanes 2-10 are genome DNA extracted from different tomato individual plants;
FIG. 3 is a 1% agarose gel electrophoresis of genomic DNA of potato leaf flakes extracted by the method of the present invention, wherein lane 1 is Marker (BM2000 DNA Marker), and lanes 2-11 are genomic DNA extracted from different potato plants;
FIG. 4 is a 1% agarose gel electrophoresis of cotton leaf genome DNA extracted by the method of the present invention, wherein lane 1 is Marker (BM5000 DNA Marker), and lanes 2-10 are genome DNA extracted from different cotton plants.
Detailed Description
The invention is further described below with reference to the figures and examples. The following examples are intended only to illustrate the invention and should not be construed as limiting it; the experimental methods in the following examples are all conventional methods; materials and reagents used in the following examples are commercially available; the instrumentation used in the examples described below is conventional in molecular biology laboratories. Modifications or substitutions to methods, steps or conditions of the invention by those skilled in the art without departing from the spirit and substance of the invention are within the scope of the invention.
Example 1: extraction of genomic DNA of Rice
The extraction method of the genome DNA of the fresh mature leaves of the rice comprises the following steps:
prior to the experiment, FN-S cell lysates were prepared: FN-S cell lysate is prepared from 1-10% (w/v) CTAB, 10-300 mM Tris-HCl (pH 7.5-8.5), and 1-50 mM Na2EDTA (pH 7.5-8.5), 1.4-2.0M NaCl, 0-2% (w/v) PVP, 0.5-2% (v/v) Triton X-100 and pure water, and any mixture ratio in the above range can achieve the same or similar effect, and in the embodiment, the concentration is 2% (w/v) CTAB, 100mM Tris-HCl (pH 8.0) and 10mM Na2EDTA (pH 8.0), 1.4M NaCl, 1% (v/v) Triton X-100, and pure water pH 7.5. The specific extraction steps are as follows:
(1) roughly shearing 50mg of fresh leaves, putting into a clean 2ml centrifuge tube with 3 steel balls (diameter is 4mm), quickly freezing in liquid nitrogen for 5s, and grinding the leaves into powder by using a tissue grinder;
(2) adding 200ul of FN-S cell lysate which is preheated at 70 ℃ for heavy suspension precipitation, adding 4ul of RNase A, oscillating up and down for 5S, and bathing at 70 ℃ for 10 min;
(3)13500rpm for 1min, and then transferring 150ul of the supernatant to a clean 1.5ml centrifuge tube;
(4) adding 150ul isopropanol (normal temperature or low temperature), shaking up and down for 5s, centrifuging at 13500rpm for 30s, and discarding the supernatant;
(5) resuspending the pellet with 400uL of 70% ethanol, shaking up and down for 5s, centrifuging at 13500rpm for 30s, and discarding the supernatant;
(6) resuspending the precipitate with 800uL of anhydrous ethanol, shaking up and down for 5s, centrifuging at 13500rpm for 30s, and discarding the supernatant;
(7) resuspending the precipitate with 800uL of anhydrous ethanol, shaking up and down for 5s, centrifuging at 13500rpm for 30s, sucking the supernatant with a suction head, and retaining the precipitate;
(8) opening the cover of the centrifuge tube with the precipitate, and placing the centrifuge tube in an air-blast drying oven at 70 ℃ for 2min for airing;
(9) resuspend the pellet with 50ul of sterile ultrapure water preheated at 70 ℃, centrifuge at 13500rpm for 30s, the supernatant is the genomic DNA solution, and transfer the supernatant to a clean centrifuge tube for preservation.
(II) detecting the quality of the genome DNA of the fresh mature leaves of the rice:
by using the method described in (a) of this example, the genomic DNA of fresh mature leaves from 10 different individual plants of nipponica rice was rapidly extracted, and the results were as follows:
furthermore, 5ul of the above genomic DNA solutions are respectively taken, 1% agarose gel is used for electrophoresis detection, the detection result is shown in figure 1, and the genomic DNA templates of 10 different rice single plant leaves can be electrophoresed to obtain clear target bands without dragging and impurity bands, so that the repeatability is good, and the effect is ideal.
Example 2: extraction of tomato genomic DNA
The method for extracting the genomic DNA of the fresh tomato leaves comprises the following steps:
prior to the experiment, FN-S cell lysates were prepared: FN-S cell lysate is prepared from 1-10% (w/v) CTAB, 10-300 mM Tris-HCl (pH 7.5-8.5), and 1-50 mM Na2EDTA (pH 7.5-8.5), 1.4-2.0M NaCl, 0-2% (w/v) PVP, 0.5-2% (v/v) Triton X-100 and pure water, and any mixture ratio in the above range can achieve the same or similar effect, and in the embodiment, 3% (w/v) CTAB, 150mM Tris-HCl (pH 8.0) and 10mM Na are set2EDTA (pH 8.0), 1.5M NaCl, 1.5% (v/v) Triton X-100, and pure water pH 7.5. The specific extraction procedure was the same as in example 1.
(II) detecting the quality of the tomato fresh leaf genome DNA:
using the method described in (a) of this example, rapid extraction of genomic DNA was performed on fresh leaves from 9 different tomato individuals, as determined by the following:
| individual plant 1 | Individual plant 2 | Individual plant 3 | Individual plant 4 | Individual plant 5 | Individual plant 6 | Individual plant 7 | Individual plant 8 | Single plant 9 | |
| OD260/OD280 | 1.82 | 1.85 | 1.80 | 1.76 | 1.78 | 1.83 | 1.81 | 1.79 | 1.75 |
| DNA yield (ug) | 11.1 | 10.4 | 12.5 | 11.8 | 12.4 | 11.3 | 9.8 | 10.3 | 9.5 |
Furthermore, 5ul of the above genomic DNA solutions are respectively taken, 1% agarose gel is used for electrophoresis detection, the detection result is shown in figure 2, and the genomic DNA templates of 9 different tomato individual plant leaves can be electrophoresed to obtain clear target bands without dragging and impurity bands, so that the repeatability is good, and the effect is ideal.
Example 3: extraction of genomic DNA from potato
The method for extracting the genomic DNA of the fresh potato leaf comprises the following steps:
prior to the experiment, FN-S cell lysates were prepared: FN-S cell lysate is prepared from 1-10% (w/v) CTAB, 10-300 mM Tris-HCl (pH 7.5-8.5), and 1-50 mM Na2EDTA (pH 7.5-8.5), 1.4-2.0M NaCl, 0-2% (w/v) PVP, 0.5-2% (v/v) Triton X-100 and pure water, and any mixture ratio in the above range can achieve the same or similar effect, and in the embodiment, 4% (w/v) CTAB, 100mM Tris-HCl (pH 8.0) and 20mM Na are set2EDTA (pH 8.0), 1.5M NaCl, 1.5% (v/v) Triton X-100, and pure water pH 7.5. The specific extraction procedure was the same as in example 1.
(II) detecting the quality of the genomic DNA of the fresh potato leaf:
using the method described in (a) of this example, rapid extraction of genomic DNA was performed on fresh leaves from 10 different potato individuals, as determined by the following:
furthermore, 5ul of the above genomic DNA solutions are respectively taken, 1% agarose gel is used for electrophoresis detection, the detection result is shown in figure 3, and the genomic DNA templates of 10 different potato single plant leaves can be electrophoresed to obtain clear target bands without dragging and impurity bands, so that the repeatability is good, and the effect is ideal.
Example 4: extraction of cotton genomic DNA
The method for extracting the genomic DNA of the fresh cotton leaf comprises the following steps:
before the experiment, FN-PW cell rinsing solution and FN-S cell lysate are prepared: the FN-PW cell rinsing liquid consists of 0.5-4% (w/v) PVP, 0-2% (w/v) L-cysteine, 0.5-4% (v/v) Triton X-100 and pure water, and the same or similar effect can be achieved by any mixture ratio combination within the value range, and the PVP is 2% (w/v), the L-cysteine is 1% (w/v), the Triton X-100 is 1.5% (v/v) and the pure water with the pH value of 7.5 are set in the embodiment; FN-S cell lysate is prepared from 1-10% (w/v) CTAB, 10-300 mM Tris-HCl (pH 7.5-8.5), and 1-50 mM Na2EDTA (pH 7.5-8.5), 1.4-2.0M NaCl, 0-2% (w/v) PVP, 0.5-2% (v/v) Triton X-100 and pure water, and any mixture ratio in the above range can achieve the same or similar effect, and in the embodiment, the concentration is 2% (w/v) CTAB, 100mM Tris-HCl (pH 8.0) and 20mM Na2EDTA (pH 8.0), 1.4M NaCl, 0.5% (w/v) PVP, 1% (v/v) Triton X-100 and pure water pH 7.5. The specific extraction steps are as follows:
(1) roughly shearing 50mg of fresh leaves, putting into a clean 2ml centrifuge tube with 3 steel balls (diameter is 4mm), quickly freezing in liquid nitrogen for 5s, and grinding the leaves into powder by using a tissue grinder;
(2) adding 1ml of FN-PW cell rinsing liquid preheated to 70 ℃ into the centrifuge tube, rinsing the FN-PW cell rinsing liquid by oscillating up and down for 30 seconds, centrifuging the FN-PW cell rinsing liquid at 12000-14000 rpm for 1min, and removing supernatant;
(3) repeating the step (2) for 5 times;
(4) adding 200ul of FN-S cell lysate which is preheated at 70 ℃ for heavy suspension precipitation, adding 4ul of RNase A, oscillating up and down for 5S, and bathing at 70 ℃ for 10 min;
(5)13500rpm for 1min, and then transferring 150ul of the supernatant to a clean 1.5ml centrifuge tube;
(6) adding 90ul of isopropanol (normal temperature or low temperature), oscillating up and down for 5s, centrifuging at 13500rpm for 30s, and discarding the supernatant;
(7) resuspending the pellet with 400ul 70% ethanol, shaking up and down for 5s, centrifuging at 13500rpm for 30s, and discarding the supernatant;
(8) resuspending the precipitate with 800ul of absolute ethanol, shaking up and down for 5s, centrifuging at 13500rpm for 30s, and discarding the supernatant;
(9) resuspending the precipitate with 800ul of absolute ethanol, shaking up and down for 5s, centrifuging at 13500rpm for 30s, sucking the supernatant with a suction head, and retaining the precipitate;
(10) opening the cover of the centrifuge tube with the precipitate, and placing the centrifuge tube in an air-blast drying oven at 70 ℃ for 2min for airing;
(11) resuspend the pellet with 50ul of sterile ultrapure water preheated at 70 ℃, centrifuge at 13500rpm for 30s, the supernatant is the genomic DNA solution, and transfer the supernatant to a clean centrifuge tube for preservation.
(II) detecting the quality of fresh leaf genome DNA of cotton:
using the method described in (a) of this example, rapid extraction of genomic DNA was performed on fresh leaves from 9 different cotton individuals, as determined by the following:
| individual plant 1 | Individual plant 2 | Individual plant 3 | Individual plant 4 | Individual plant 5 | Individual plant 6 | Individual plant 7 | Individual plant 8 | Single plant 9 | |
| OD260/OD280 | 1.91 | 1.93 | 1.89 | 1.94 | 1.83 | 1.90 | 1.85 | 1.80 | 1.88 |
| DNA yield (ug) | 13.8 | 14.5 | 13.2 | 10.9 | 12.6 | 15.4 | 11.4 | 12.5 | 13.9 |
Furthermore, 5ul of the above genomic DNA solutions are respectively taken, 1% agarose gel is used for electrophoresis detection, the detection result is shown in figure 4, and the genomic DNA templates of 9 different cotton single plant leaves can be electrophoresed to obtain clear target bands without dragging and impurity bands, so that the repeatability is good, and the effect is ideal.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, but rather as the intention of all modifications, equivalents, improvements, and equivalents falling within the spirit and scope of the invention.
Claims (9)
1. A non-toxic plant universal high-purity genome DNA rapid extraction method comprises the following steps:
(1) grinding and crushing a plant sample to be extracted;
(2) adding FN-PW cell rinsing liquid to rinse and remove impurities such as polysaccharide polyphenol, pigment and the like in a sample, wherein the FN-PW cell rinsing liquid consists of 0.5-4% (w/v) PVP, 0-2% (w/v) L-cysteine, 0.5-4% (v/v) Triton X-100 and pure water;
(3) adding FN-S cell lysate to release genome DNA, centrifuging and taking supernatant, wherein the FN-S cell lysate is prepared from 1-10% (w/v) CTAB, 10-300 mM Tris-HCl (pH 7.5-8.5) and 1-50 mM Na2EDTA (the pH value is 7.5-8.5), 1.4-2.0M NaCl, 0-2% (w/v) PVP, 0.5-2% (v/v) Triton X-100 and pure water;
(4) adding isopropanol into the supernatant obtained in the step (3) for heavy suspension precipitation, oscillating, centrifuging and removing the supernatant;
(5) adding 70-75% ethanol for heavy suspension precipitation, oscillating, centrifuging and discarding supernatant;
(6) adding absolute ethyl alcohol to resuspend and precipitate, oscillating, centrifuging and removing supernate;
(7) drying the centrifugal precipitate obtained in the step (6), adding sterile ultrapure water or TE for heavy suspension precipitation, centrifuging and taking supernatant fluid to obtain a high-purity genome DNA solution;
wherein, the step (2) is an optional step, and can be omitted for samples containing less impurities; for samples rich in impurities such as polysaccharide, polyphenol and pigment, this step cannot be omitted; in the step (3), whether RNase A is added or not can be selected according to the purpose of an experiment when the genome DNA is released, and the RNase A is not required to be added when the obtained genome DNA is only used for a short fragment PCR amplification template below 2 kb; the obtained genomic DNA is used for cloning short fragment genes of 2kb or less, and is added with RNase A when used for downstream molecular biology experiments such as enzyme digestion, library construction, Real-time PCR and Southern Blot.
2. The method of claim 1, wherein the FN-PW cell rinse added rinse in step (2) is rinsed as follows: adding a preheated FN-PW cell rinsing solution at 50-75 ℃ into the crushed plant sample to be extracted according to the dosage of 0.02ml/mg (based on the weight of the plant sample to be extracted), shaking and rinsing up and down for 30 seconds, centrifuging at 12000-14000 rpm for 1min, and discarding the supernatant.
3. The method according to claim 1, wherein when the obtained genomic DNA is used only for PCR amplification of a template for a short fragment of 2kb or less, and when the genomic DNA is released in the step (3), the release of the genomic DNA is carried out as follows: adding FN-S cell lysate which is preheated to 50-75 ℃ according to the dosage of 4ul/mg (based on the weight of a plant sample to be extracted) for heavy suspension precipitation, oscillating up and down for 5S, and bathing for 2-5 min at 50-75 ℃; when the obtained genomic DNA is used for downstream molecular biology experiments such as enzyme digestion, library construction, Real-time PCR and Southern Blot as well as for cloning of the following short-fragment genes of 2kb, the genomic DNA is released in the following manner when the genomic DNA is released in step (3): adding FN-S cell lysate which is preheated at 50-75 ℃ according to the dosage of 4ul/mg (based on the weight of a plant sample to be extracted) for resuspending precipitation, adding RNase A according to the dosage of 1:0.02(v/v, based on the dosage of the FN-S cell lysate), oscillating the mixture up and down for 5S, and carrying out warm bath at 50-75 ℃ for 5-15 min.
4. The method according to claim 1, wherein the isopropanol is added in the step (4) to resuspend the precipitate in an amount of 0.6 to 1 times the volume of the supernatant obtained in the step (3).
5. The method according to claim 1, wherein when 70-75% of ethanol is added in the step (5) for resuspension of the precipitate, 70-75% of ethanol is added in an amount of 8-16 ul/mg (based on the weight of the plant sample to be extracted) for resuspension of the precipitate.
6. The method according to claim 1, wherein when the absolute ethyl alcohol is added in the step (6) for resuspending the precipitate, the absolute ethyl alcohol is added in an amount of 8-16 ul/mg (based on the weight of the plant sample to be extracted).
7. The method according to claim 1, wherein the centrifugal precipitate obtained in the step (6) is dried in the step (7) according to the following method: and opening the cover of the centrifuge tube with the precipitate, and placing the centrifuge tube in a forced air drying oven at 65-75 ℃ for airing for 1-5 min.
8. The method of claim 1, wherein the resuspension of the pellet is performed with preheated sterile ultrapure water or TE in the step (7) when adding the sterile ultrapure water or TE.
9. A non-toxic plant-wide rapid extraction kit for high-purity genomic DNA, comprising the FN-PW cell rinse of claim 1 or the FN-S cell lysate of claim 1 or both the FN-PW cell rinse of claim 1 and the FN-S cell lysate of claim 1.
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