Plant genomic dna extraction reagent

Abstract

The application discloses a plant genome DNA extraction reagent, which comprises Buffer 1 lysate, Buffer 2 precipitate and Buffer 3 combination liquid; the Buffer 1 lysate comprises sodium chloride, PVP40, sodium metabisulfite and SDS; wherein the concentration of the sodium chloride is 250-1200 mM; the mass percentage of the PVP40 is 0.5%-3%; the concentration of the sodium metabisulfite is 50-150 mM; and the mass percentage of the SDS is 0.7%-2.5%; the Buffer 2 precipitate comprises acetate; wherein the concentration of the acetate is 2.2-4.3 M; and the Buffer 3 combination liquid comprises guanidine hydrochloride; wherein the concentration of the guanidine hydrochloride is 0.5-2.5 M. The extraction reagent does not contain organic solvents such as phenol and chloroform, and can improve the removal rate of polyphenols.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, more particularly, it relates to a plant genomic DNA extraction reagent. BACKGROUND

[0002] Isolation of high quality genomic DNA (gDNA) is a critical step in modern molecular biology research. Many methods have been developed for extracting gDNA from various plant species. Many plants contain abundant secondary metabolites (such as polysaccharides and polyphenols). Plant tissues and cells produce secondary metabolites such as phenolic compounds and polysaccharides, which often interfere with DNA isolation and its application in downstream techniques. These secondary plant metabolites can destroy the purification of DNA and / or degrade it in most conventional plant DNA.

[0003] Phenol-chloroform separation is a common method in many genomic DNA isolation protocols, phenol-chloroform is a commonly used organic reagent for removing proteins from DNA samples. However, phenol-chloroform is harmful to human health and the environment. Special operating and personal protective equipment must be used when handling phenol-chloroform. In addition, phenol inhibits enzymatic reactions in downstream applications. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides a plant genomic DNA extraction reagent, which does not contain organic solvents such as phenol and chloroform, does not contain compounds such as B-mercaptoethanol, and can improve the removal rate of polyphenols.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0006] The plant genomic DNA extraction reagent comprises Buffer 1 lysis solution, Buffer 2 precipitation solution and Buffer 3 binding solution;

[0007] The Buffer 1 lysis solution comprises sodium chloride, PVP40, metabisulfite sodium and SDS; wherein the concentration of sodium chloride is 250-1200mM; the mass percentage of PVP40 is 0.5%-3%; the concentration of metabisulfite sodium is 50-150mM; the mass percentage of SDS is 0.7%-2.5%;

[0008] The Buffer 2 precipitation solution comprises acetate; wherein the concentration of acetate is 2.2-4.3M;

[0009] The Buffer 3 binding solution comprises guanidine hydrochloride; wherein the concentration of guanidine hydrochloride is 0.5-2.5M.

[0010] In summary, the present application has the following beneficial effects:

[0011] 1. The present invention discloses a solution required for extracting plant genomic DNA, which is clear in composition and contains non-organic solvents, to extract microgram (1-13 μg) gDNA from fresh plant tissues in 96-well plates at low cost, and the scheme is effective for several easy and difficult-to-extract forage grasses, crops, gardening and common model species, including barley leaves, wheat leaves, rice, potatoes and Arabidopsis thaliana.

[0012] 2. The DNA extracted by the extraction reagent of the present invention has high molecular weight and is easy to digest with restriction enzymes; the scheme is scalable and easy to automate, does not require a fume hood, and takes about three hours to process 192 samples, and the consumable price is low, at $0.62 per sample;

[0013] 3. The multifunctional, scalable and simple scheme of the present invention can produce high-molecular-weight genomic DNA suitable for restriction enzyme digestion and next-generation sequencing applications, including GBS and long-read sequencing platforms (such as PacBio); the method is low in cost and high in throughput, and can extract high-quality gDNA from various fresh and dry plant source materials, suitable for many sequencing and genotyping applications, including large-scale sample screening to support breeding programs. DETAILED DESCRIPTION

[0014] A reagent for extracting plant genomic DNA does not contain organic solvents such as phenol and chloroform, and does not contain compounds such as B-mercaptoethanol, uses Pall AcroPrep TM Advance 96-well 1-milliliter filter plate instead of a separate silica gel filter cartridge spin column; the same plate can be reused for multiple individual extractions, but a fresh well is used for each extraction.

[0015] The extraction method includes grinding the tissue into powder with a tissue grinder and adding it to the lysis solution and mixing; adding the precipitation solution, incubating at room temperature or 65°C for 10 minutes, centrifuging to obtain the supernatant, adding the binding solution to the 96-well filter plate to adsorb the DNA on the membrane, rinsing twice with the rinse solution, and then centrifuging to obtain the genomic DNA.

[0016] The lysis solution contains sodium chloride, Tris-Hcl, PVP40, metabisulfite sodium, SDS and isopropyl alcohol; the precipitation solution contains acetate, and the binding solution containing guanidine hydrochloride and ethanol is added to the above supernatant for nucleic acid adsorption, the adsorbed membrane is washed twice with the rinse solution and then centrifuged, and the genomic DNA is dissolved with the dissolution solution. Among them, the plant grinding needs to be in the environment of liquid nitrogen.

[0017] In the Buffer 1 lysis solution, the Buffer 1 lysis solution comprises sodium chloride, Tris-Hcl, PVP40, sodium metabisulfite, SDS and isopropyl alcohol; the concentration of sodium chloride is 250-1000 mM; the concentration of Tris-Hcl is 50-100 mM; the mass percentage of PVP40 is 0.5%-3%; the concentration of sodium metabisulfite is 50-150 mM; the mass percentage of SDS is 0.7%-2.5%; the volume percentage of isopropyl alcohol is 5%-10%; and the pH of the lysis solution is 6.5-8.

[0018] In the Buffer 2 precipitation solution, the acetate can be one of sodium acetate and potassium acetate, and the concentration of the acetate is 3-3.6 M; the concentration of acetic acid is 2-3.2 M.

[0019] In the Buffer 3 binding solution, the concentration of guanidine hydrochloride is 1-2.5 M, and the Buffer 3 binding solution is configured by a mixture of Tris-Hcl and EDTA, wherein the volume ratio of Tris-Hcl to EDTA is (10-15):(1-2).

[0020] The supernatant after sample lysis and precipitation needs to be transferred to an adsorption plate and 1.5 times of the binding solution is added; the sample after lysis and precipitation can be placed at room temperature or 65°C for 10 minutes and centrifuged at the maximum centrifugal force for 5-10 minutes; the concentration of ethanol in the rinsing solution is 70%-85%; and the elution solution has a pH of 7-8.

[0021] The plants include potatoes, barley, wheat and Arabidopsis thaliana.

[0022] Example 1: Taking potatoes as samples, high-quality genomic DNA is extracted.

[0023] The extraction reagent comprises the following components in mass fraction or volume fraction:

[0024] The Buffer 1 lysis solution: 500 mM sodium chloride, 100 mM Tris-Hcl (pH 7.4), 1% PVP40, 100 mM sodium metabisulfite, 2% SDS, 10% isopropyl alcohol, 50 mM EDTA;

[0025] The Buffer 2 precipitation solution: 3.6 M potassium acetate, 2.4 M acetic acid;

[0026] The Buffer 3 binding solution: 2 M guanidine hydrochloride (1000 ml is prepared, 191 g of guanidine hydrochloride is added, and the solution is prepared to 333 ml by using TE (Tris EDTA 10:1, pH 8), and 667 ml of anhydrous ethanol is added);

[0027] Buffer 4 rinse solution: To prepare 1000ml, add 2ml 1MTris-HCl (pH 8) and 800ml anhydrous ethanol, and make up the rest to 1000ml with ddH2O;

[0028] Buffer 5: 10mM Tris-HCl (pH 8), 1mM EDTA

[0029] Cell lysis: Place 50 mg of fresh tissue and two 5 / 32 inch (3.97 mm) stainless steel beads in a 1 ml per well of a deep-well 96-well plate. Heat seal using a thermal adhesive seal, which better prevents the beads from breaking through the seal during grinding, and then float the plate in liquid nitrogen for 5 minutes. Similarly, pre-chill the Qiagen TissueLyser plate adapter. Place the 96-well plate into the Qiagen TissueLyser plate adapter and grind the tissue at 30 Hz for 1 minute, then repeat after changing the orientation of the plate in the adapter for more uniform grinding. If homogenizing only one plate at a time, use a second balanced plate. Centrifuge at 4000 × g for 1 minute at -15°C in a Hettich Rotanta 460R centrifuge (or similar) with a swinging rotor to allow the plant material to settle to the bottom of the wells. Carefully remove the seal and cover the well with 400 μl of Buffer 1 lysis buffer and 6 ul of RNase (10 mg / ml) from the reservoir using a multichannel pipette. Mix the seal thoroughly by shaking it up and down manually (do not use a vortexer).

[0030] Nucleic acid purification and extraction: Add 130ul Buffer 2 precipitation solution to the above treated solution and cover Place the sealing pad at room temperature for 10 minutes, then centrifuge at maximum centrifugal force for 10 minutes at room temperature. Take the supernatant from the previous step, calculate the volume of the supernatant, and add 1.5 times the volume of the supernatant buffer 3 binding solution. Gently pipette up and down 10 times to mix thoroughly. Transfer all the liquid (including the precipitate) to the Pall AcroPrep TM Advance 96-well 1ml filter plate and place 1.1ml 96-well deep-well plate, centrifuge at 4000×g for 2 minutes at room temperature. Discard the flow-through and wipe the surface of the collection plate with a paper towel. Wash each well with 300μl of Buffer 4. Centrifuge at 4000×g for 2 minutes at room temperature, discard the flow-through, repeat this step once, discard the flow-through, centrifuge at 4000×g for 2 minutes at room temperature to dry the membrane, and replace it with a fresh one. 1. 1 ml 96-well deep well plate for collection of DNA to be eluted from filter plate. Add 50 μΐ Buffer 5 lysis solution to each well, let stand for 1 min, then centrifuge at 4000 x g for 1 min at room temperature to obtain genomic extract.

[0031] Genomic extract was measured using Thermo fisher nanodrop and results are shown in Table 1.

[0032] Example 2: High quality genomic DNA was extracted using barley leaves as sample.

[0033] The extraction reagents included the following components in mass or volume fraction:

[0034] Buffer 1 lysis solution: 500 mM sodium chloride, 100 mM Tris-Hcl (pH 7.4), 1% PVP40, 2% SDS, 10% isopropanol, 50 mM EDTA;

[0035] Buffer 2 precipitation solution: 3.6 M potassium acetate, 2.4 M acetic acid;

[0036] Buffer 3 binding solution: 2 M guanidine hydrochloride (to make 1000 ml, add 191 g guanidine hydrochloride, make up to 333 ml with TE (Tris EDTA 10: 1, pH 8), add 667 ml absolute ethanol);

[0037] Buffer 4 rinse solution: to make 1000 ml, add 2 ml 1 M Tris-Hcl (pH 8), 800 ml absolute ethanol, make up to 1000 ml with ddH20;

[0038] Buffer 5 lysis solution: 10 mM Tris-Hcl (pH 8), 1 mM EDTA;

[0039] Cell lysis: 50 mg of fresh tissue and two 5 / 32 inch (3.97 mm) stainless steel beads were placed in a 2 ml microcentrifuge tube. 1 ml per well of a deep-well 96-well plate. Heat seal using a thermal adhesive seal, which better prevents the beads from breaking through the seal during grinding, and then float the plate in liquid nitrogen for 5 minutes. Similarly, pre-chill the Qiagen TissueLyser plate adapter. Place the 96-well plate into the Qiagen TissueLyser plate adapter and grind the tissue at 30 Hz for 1 minute, then repeat after changing the orientation of the plate in the adapter for more uniform grinding. If homogenizing only one plate at a time, use a second balanced plate. Centrifuge at 4000 × g for 1 minute at -15°C in a Hettich Rotanta 460R centrifuge (or similar) with a swinging rotor to allow the plant material to settle to the bottom of the wells. Carefully remove the seal and cover the well with 400 μl of Buffer 1 lysis buffer and 6 ul of RNase (10 mg / ml) from the reservoir using a multichannel pipette. Mix the seal thoroughly by shaking it up and down manually (do not use a vortexer).

[0040] Nucleic acid purification and extraction: Add 130ul Buffer 2 precipitation solution to the above treated solution and cover Place the sealing pad at room temperature for 10 minutes, then centrifuge at maximum centrifugal force for 10 minutes at room temperature. Take the supernatant from the previous step, calculate the volume of the supernatant, and add 1.5 times the volume of the supernatant buffer 3 binding solution. Gently pipette up and down 10 times to mix thoroughly. Transfer all the liquid (including the precipitate) to the Pall AcroPrep TM Advance 96-well 1ml filter plate and place 1.1ml 96-well deep-well plate, centrifuge at 4000×g for 2 minutes at room temperature. Discard the flow-through and wipe the surface of the collection plate with a paper towel. Wash each well with 300μl of Buffer 4. Centrifuge at 4000×g for 2 minutes at room temperature, discard the flow-through, repeat this step once, discard the flow-through, centrifuge at 4000×g for 2 minutes at room temperature to dry the membrane, and replace it with a fresh one. A 1.1ml 96-well deep-well plate is used to collect DNA to be eluted from the filter plate. Add 50 μl of Buffer 5 to each well, let it stand for 1 minute, and then centrifuge at 4000 × g for 1 minute at room temperature to obtain the genomic extract.

[0041] The results of the genomic extracts measured using the Thermo Fisher nanodrop are shown in Table 1 .

[0042] Example 3: Extracting high-quality genomic DNA from wheat leaves.

[0043] The extraction reagent includes the following components in mass fraction or volume fraction:

[0044] Buffer 1 lysis buffer: 500 mM NaCl, 100 mM Tris-HCl (pH 7.4), 1% PVP40, 100 mM sodium metabisulfite, 2% SDS, 10% isopropanol, 50 mM EDTA;

[0045] Buffer 2 precipitation solution: 3.6 M sodium acetate, 2.4 M acetic acid;

[0046] Buffer 3 binding solution: 2 M guanidine hydrochloride (to prepare 1000 ml, add 191 g guanidine hydrochloride, make up to 333 ml with TE (Tris EDTA 10:1, pH 8), and add 667 ml of anhydrous ethanol);

[0047] Buffer 4 rinse solution: To prepare 1000ml, add 2ml 1MTris-HCl (pH 8) and 800ml anhydrous ethanol, and make up the rest to 1000ml with ddH2O;

[0048] Buffer 5: 10 mM Tris-HCl (pH 8), 1 mM EDTA;

[0049] Cell lysis: Place 50 mg of fresh tissue and two 5 / 32 inch (3.97 mm) stainless steel beads in a 1 ml per well of a deep-well 96-well plate. Heat seal using a thermal adhesive seal, which better prevents the beads from breaking through the seal during grinding, and then float the plate in liquid nitrogen for 5 minutes. Similarly, pre-chill the Qiagen TissueLyser plate adapter. Place the 96-well plate into the Qiagen TissueLyser plate adapter and grind the tissue at 30 Hz for 1 minute, then repeat after changing the orientation of the plate in the adapter for more uniform grinding. If homogenizing only one plate at a time, use a second balanced plate. Centrifuge at 4000 × g for 1 minute at -15°C in a Hettich Rotanta 460R centrifuge (or similar) with a swinging rotor to allow the plant material to settle to the bottom of the wells. Carefully remove the seal and cover the well with 400 μl of Buffer 1 lysis buffer and 6 ul of RNase (10 mg / ml) from the reservoir using a multichannel pipette. Mix the seal thoroughly by shaking it up and down manually (do not use a vortexer).

[0050] Nucleic acid purification and extraction: Add 130ul Buffer 2 precipitation solution to the above treated solution and cover The sealant was left at room temperature for 10 minutes and then centrifuged at maximum centrifugal force for 10 minutes at room temperature. The supernatant from the previous step was taken and the volume of the supernatant was calculated, 1.5 times the volume of the supernatant was added to the Buffer 3 binding solution. Pipette up and down 10 times gently, mix well. Move all the liquid (including the precipitate) into a Pall AcroPrep TM Advance 96-well 1 ml filter plate and placed in 1.1 ml 96-well deep well plate, centrifuged at 4000 x g for 2 minutes at room temperature. Discard the effluent and wipe the surface of the collection plate with a paper towel, rinse each well with 300 μl of Buffer 4 rinse solution. Centrifuge at 4000 x g for 2 minutes at room temperature, discard the effluent, repeat this step once, discard the effluent, centrifuge at 4000 x g for 2 minutes at room temperature, replace with a dry membrane, a new 1.1 ml 96-well deep well plate for collecting the DNA to be eluted from the filter plate. Add 50 μl of Buffer 5 lysis solution to each well, centrifuge at 4000 x g for 1 minute at room temperature after standing for 1 minute to obtain the genomic extract.

[0051] The genomic extract was measured using a Thermo fisher nanodrop and the results are shown in Table 1.

[0052] Example 4: High quality genomic DNA was extracted using tomato leaves as the sample.

[0053] The extraction reagent includes the following components in mass fraction or volume fraction:

[0054] Buffer 1 lysis solution: 1000 mM sodium chloride, 100 mM Tris-Hcl (pH 7.4), 1% PVP40, 100 mM sodium metabisulfite, 2% SDS, 10% isopropyl alcohol, 50 mM EDTA;

[0055] Buffer 2 precipitation solution: 3.6 M potassium acetate, 2.4 M acetic acid;

[0056] Buffer 3 binding solution: 2 M guanidine hydrochloride (prepare 1000 ml, add 191 g guanidine hydrochloride, prepare to 333 ml with TE (Tris EDTA 10: 1, pH 8), add 667 ml of absolute ethanol);

[0057] Buffer 4 rinse solution: prepare 1000 ml, add 2 ml of 1 M Tris-Hcl (pH 8), 800 ml of absolute ethanol, and the rest is made up to 1000 ml with ddH2O;

[0058] Buffer 5 lysis solution: 10 mM Tris-Hcl (pH 8), 1 mM EDTA;

[0059] Cell lysis: Place 50 mg of fresh tissue and two 5 / 32 inch (3.97 mm) stainless steel beads in a 1 ml per well of a deep-well 96-well plate. Heat seal using a thermal adhesive seal, which better prevents the beads from breaking through the seal during grinding, and then float the plate in liquid nitrogen for 5 minutes. Similarly, pre-chill the Qiagen TissueLyser plate adapter. Place the 96-well plate into the Qiagen TissueLyser plate adapter and grind the tissue at 30 Hz for 1 minute, then repeat after changing the orientation of the plate in the adapter for more uniform grinding. If homogenizing only one plate at a time, use a second balanced plate. Centrifuge at 4000 × g for 1 minute at -15°C in a Hettich Rotanta 460R centrifuge (or similar) with a swinging rotor to allow the plant material to settle to the bottom of the wells. Carefully remove the seal and cover the well with 400 μl of Buffer 1 lysis buffer and 6 ul of RNase (10 mg / ml) from the reservoir using a multichannel pipette. Mix the seal thoroughly by shaking it up and down manually (do not use a vortexer).

[0060] Nucleic acid purification and extraction: Add 130ul Buffer 2 precipitation solution to the above treated solution and cover Place the sealing pad at room temperature for 10 minutes, then centrifuge at maximum centrifugal force for 10 minutes at room temperature. Take the supernatant from the previous step, calculate the volume of the supernatant, and add 1.5 times the volume of the supernatant buffer 3 binding solution. Gently pipette up and down 10 times to mix thoroughly. Transfer all the liquid (including the precipitate) to the Pall AcroPrep TM Advance 96-well 1ml filter plate and place 1.1ml 96-well deep-well plate, centrifuge at 4000×g for 2 minutes at room temperature. Discard the flow-through and wipe the surface of the collection plate with a paper towel. Wash each well with 300μl of Buffer 4. Centrifuge at 4000×g for 2 minutes at room temperature, discard the flow-through, repeat this step once, discard the flow-through, centrifuge at 4000×g for 2 minutes at room temperature to dry the membrane, and replace it with a fresh one. A 1.1ml 96-well deep-well plate is used to collect DNA to be eluted from the filter plate. Add 50 μl of Buffer 5 to each well, let it stand for 1 minute, and then centrifuge at 4000 × g for 1 minute at room temperature to obtain the genomic extract.

[0061] The results of the genomic extracts measured using the Thermo Fisher nanodrop are shown in Table 1 .

[0062] Example 5: Using Arabidopsis thaliana as a sample, high-quality genomic DNA was extracted.

[0063] The extraction reagent includes the following components in mass fraction or volume fraction:

[0064] Buffer 1 lysis buffer: 500 mM NaCl, 100 mM Tris-HCl (pH 7.4), 1% PVP40, 100 mM sodium metabisulfite, 2% SDS, 10% isopropanol, 50 mM EDTA;

[0065] Buffer 2 precipitation solution: 3.6 M potassium acetate, 2.4 M acetic acid;

[0066] Buffer 3 binding solution: 1 M guanidine hydrochloride (to prepare 1000 ml, add 95.5 g guanidine hydrochloride, make up to 333 ml with TE (Tris EDTA 10:1, pH 8), and add 667 ml of anhydrous ethanol);

[0067] Buffer 4 rinse solution: To prepare 1000ml, add 2ml 1MTris-HCl (pH 8) and 800ml anhydrous ethanol, and make up the rest to 1000ml with ddH2O;

[0068] Buffer 5: 10 mM Tris-HCl (pH 8), 1 mM EDTA;

[0069] Cell lysis: Place 50 mg of fresh tissue and two 5 / 32 inch (3.97 mm) stainless steel beads in a 1ml per well of a deep-well 96-well plate. Heat seal using a thermal adhesive seal, which better prevents the beads from breaking through the seal during grinding, and then float the plate in liquid nitrogen for 5 minutes. Similarly, pre-cool the Qiagen TissueLyser plate adapter. Place the 96-well plate into the Qiagen TissueLyser plate adapter and grind the tissue at 30Hz for 1 minute, then repeat the above operation after changing the orientation of the plate in the adapter for more uniform grinding. If homogenizing only one plate at a time, use a second balanced plate. Centrifuge at 4000×g for 1 minute at -15°C using a Hettich Rotanta 460R centrifuge (or similar equipment) with a swinging rotor to allow the plant material to settle to the bottom of the wells. Carefully remove the seal and add 400μl of Buffer 1 lysis solution and 6ul RNase (10mg / ml) from the reservoir to each well using a multichannel pipette and cover. Mix the seal thoroughly by shaking it up and down manually (do not use a vortexer).

[0070] Nucleic acid purification and extraction: To the above treated solution, 130ul Buffer 2 precipitation solution was added, and the lid was covered The sealing pad was placed at room temperature for 10 minutes, and then centrifuged at maximum centrifugal force for 10 minutes at room temperature. The supernatant of the previous step was taken, the volume of the supernatant was calculated, and 1.5 times the volume of the supernatant was added. Buffer 3 binding solution. Pipette up and down 10 times, mix well. Move all the liquid (including the precipitate) into the Pall AcroPrep TM Advance 96-well 1ml filter plate and placed in 1.1ml 96-well deep well plate, centrifuged at 4000xg for 2 minutes at room temperature. Discard the effluent and wipe the surface of the collection plate with a paper towel, rinse each well with 300ul Buffer 4 rinse solution. Centrifuge at 4000xg for 2 minutes at room temperature, discard the effluent, repeat this step once, discard the effluent, centrifuge at 4000xg for 2 minutes at room temperature, replace with a new 1.1ml 96-well deep well plate, used to collect the DNA to be eluted from the filter plate. Add 50ul Buffer 5 dissolution solution to each well, centrifuge at 4000xg for 1 minute at room temperature to obtain the genomic extract.

[0071] The genomic extract was measured by Thermo fisher nanodrop, and the results are shown in Table 1.

[0072] Example 6: The difference between this example and Example 1 is that the nucleic acid extraction and purification: To the above treated solution, 130ul Buffer 2 precipitation solution was added, and the lid was covered The sealing pad was placed at room temperature for 10 minutes, and then centrifuged at maximum centrifugal force for 10 minutes at room temperature. The supernatant of the previous step was taken, the volume of the supernatant was calculated, and 1.5 times the volume of the supernatant was added. Buffer 3 binding solution. Pipette up and down 10 times, mix well. Move all the liquid (including the precipitate) into the Pall AcroPrep

[0073] Example 7: The difference between this example and Example 1 is that 2% SDS in Buffer 1 lysis solution is changed to 1% SDS, and the genomic extract is measured by Thermo fisher nanodrop, and the results are shown in Table 1.

[0074] Example 8: The difference between this example and Example 1 is that Buffer 1 lysis solution is changed to 50mM metabisulfite sodium, and the genomic extract is measured by Thermo fisher nanodrop, and the results are shown in Table 1.

[0075] Example 9: The difference between this example and Example 1 is that Buffer 1 lysis solution is changed to 0.5% PVP40, and the genomic extract is measured by Thermo fisher nanodrop, and the results are shown in Table 1.

[0076] Table 1

[0077]

[0078]

[0079] Note: When measured using a Thermo Fisher Nanodrop UV spectrophotometer, the OD260 / OD280 ratio is generally between 1.7-1.9, and the OD260 / OD230 ratio is between 2.0-2.5. If buffer 5 is not used for elution and ddH2O is used instead, the ratio will be lower because the pH value and the presence of ions will affect the absorbance value, but it does not mean that the purity is low.

[0080] As can be seen from the results of Examples 1 to 9, Table 1 shows that the genomic DNA extracted from the lysate, precipitate, and binding solution used in the present invention is of high quality; Examples 1 and 2 show that the dual components of PVP40 and sodium metabisulfite can improve the removal rate of polyphenols, but neither can be achieved alone; Examples 1 and 3 show that potassium acetate is more capable of precipitating and removing polysaccharides from the genome than sodium acetate; Examples 1 and 4 show that 500mM sodium chloride contains less RNA residue than 1000mM sodium chloride; Examples 1 and 5 show that 2M guanidine hydrochloride has a higher binding rate than 1M guanidine hydrochloride; Examples 1 and 6 show that 130ul of Buffer 2 precipitate was added to the treated solution, and the solution was covered with a The genome yield was higher when the sealing pad was placed in a 65°C water bath for 10 minutes after leaving it in the water for 10 minutes at room temperature. Examples 1 and 7 show that 2% SDS is more effective than 1% SDS for lysis. Therefore, Example 6 achieved the best results, with the highest genome concentration extracted.

[0081] The comparison between Example 8 and Example 9 and Example 1 shows that reducing the concentration of metabisulfite and PVP40 has an impact on the concentration and purity of the experimental results; the plant genomic DNA extracted in Example 1 and Example 6 is suitable for restriction enzyme digestion and next-generation sequencing applications, including GBS and long-read sequencing platforms (such as PacBio).

[0082] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. Plant genomic DNA extraction reagent, characterized by: Using PallAcroPrep TM Advance 96-well 1 ml filter plates, including Buffer 1 lysis buffer, Buffer 2 precipitation buffer, and Buffer 3 binding buffer; The Buffer 1 lysis solution includes sodium chloride, PVP40, sodium metabisulfite and SDS; wherein the concentration of sodium chloride is 250-1200 mM; the mass percentage of PVP40 is 0.5%-3%; the concentration of sodium metabisulfite is 50-150 mM; the mass percentage of SDS is 0.7%-2.5%; The Buffer 2 precipitation solution includes acetate and acetic acid; wherein the acetate includes potassium acetate or sodium acetate, and the concentration of the acetate is 3-3.6M; the concentration of the acetic acid is 2-3.2M; The Buffer 3 binding solution includes guanidine hydrochloride; wherein the concentration of guanidine hydrochloride is 0.5-2.5M.

2. The plant genomic DNA extraction reagent according to claim 1, characterized in that: In the Buffer 1 lysis solution, the concentration of sodium chloride is 500-1000 mM.

3. The plant genomic DNA extraction reagent according to claim 2, characterized in that: In the Buffer 1 lysis solution, the concentration of PVP40 is 1% to 2%.

4. The plant genomic DNA extraction reagent according to claim 3, characterized in that: In the Buffer 1 lysis solution, the mass percentage of SDS is 1% to 2%.

5. The plant genomic DNA extraction reagent according to claim 4, characterized in that: The Buffer 1 lysis buffer comprises sodium chloride, Tris-HCl, PVP40, sodium metabisulfite, SDS, and isopropanol; the concentration of sodium chloride is 500-1000 mM; the concentration of Tris-HCl is 50-100 mM; the mass percentage of PVP40 is 1-2%; and the concentration of sodium metabisulfite is 50-100 mM. The mass percentage of the SDS is 1-2%; the volume percentage of the isopropyl alcohol is 5-10%.

6. The plant genomic DNA extraction reagent according to claim 1, characterized in that: In the Buffer 3 binding solution, the concentration of guanidine hydrochloride is 1-2.5M.

7. The plant genomic DNA extraction reagent according to claim 6, characterized in that: The Buffer 3 binding solution is prepared using a mixture of Tris-HCl and EDTA, wherein the volume ratio of Tris-HCl to EDTA is (10-15): (1-2).

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