Plant genome DNA extraction reagent

Abstract

The invention discloses a plant genome DNA (deoxyribonucleic acid) extraction reagent. The plant genome DNA extraction reagent comprises a Buffer 1 lysis solution, a Buffer 2 precipitation solution and a Buffer 3 binding solution, the Buffer 1 lysate comprises sodium chloride, PVP40 (Polyvinyl Pyrrolidone 40), sodium metabisulfite and SDS (Sodium Dodecyl Sulfate); wherein the concentration of the sodium chloride is 250 to 1200 mM; the mass percent of the PVP40 is 0.5%-3%; the concentration of the sodium metabisulfite is 50-150mM; the mass percent of the SDS is 0.7%-2.5%; the Buffer 2 precipitation solution comprises acetate; wherein the concentration of the acetate is 2.2 to 4.3 M; the Buffer 3 binding solution comprises guanidine hydrochloride; wherein the concentration of the guanidine hydrochloride is 0.5 to 2.5 M. The extraction reagent does not contain organic solvents such as phenol and chloroform, and the removal rate of polyphenol can be improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a reagent for extracting plant genomic DNA. Background Art

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

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

[0004] In view of the shortcomings of the prior art, the present invention 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 object, the present invention provides the following technical solutions:

[0006] Plant genomic DNA extraction reagents, including Buffer 1 lysis solution, Buffer 2 precipitation solution and Buffer 3 binding solution;

[0007] 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%;

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

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

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

[0011] 1. The present invention discloses a ready-to-use plant genomic DNA extraction solution, which has a clear composition and contains a non-organic solvent, and can be used to extract micrograms (1-13 μg) of gDNA from fresh plant tissues in a 96-well plate at low cost. The solution is effective for several easy and difficult to extract forage, crops, horticulture and common model species, including barley leaves, wheat leaves, rice, potatoes and Arabidopsis;

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

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

[0015] A reagent for extracting plant genomic DNA, which does not contain organic solvents such as phenol and chloroform, and does not contain compounds such as B-mercaptoethanol. TM Advance 96-well 1 ml filter plates instead of individual silica-filter spin columns; the same plate can be reused for multiple separate extractions, but fresh wells are used for each extraction.

[0016] The extraction method includes grinding the tissue into powder with a tissue grinder and adding it to the lysis solution and mixing; adding the precipitant solution, incubating at room temperature or 65°C for 10 minutes, obtaining the supernatant after centrifugation, adding the binding solution, and adsorbing the DNA on the membrane through a 96-well filter plate, rinsing twice with the rinse solution and then drying, and adding the dissolving solution to obtain the genomic DNA.

[0017] The lysis solution contains sodium chloride, Tris-Hcl, PVP40, sodium metabisulfite, SDS and isopropanol; the precipitation solution contains acetate, and a binding solution containing guanidine hydrochloride and ethanol is added to the above supernatant for nucleic acid adsorption. The adsorption membrane is washed twice with a rinse solution and then dried, and the genomic DNA is dissolved with a dissolving solution. Among them, plant grinding needs to be in a liquid nitrogen environment.

[0018] In Buffer 1 lysis buffer, Buffer 1 lysis buffer includes sodium chloride, Tris-Hcl, PVP40, sodium metabisulfite, SDS and isopropanol; the concentration of sodium chloride is 250-1000mM; the concentration of Tris-Hcl is 50-100mM; the mass percentage of PVP40 is 0.5%-3%; the concentration of sodium metabisulfite is 50-150mM; the mass percentage of SDS is 0.7%-2.5%; the volume percentage of isopropanol is 5%-10%; and the pH of the lysis buffer is 6.5-8.

[0019] In the Buffer 2 precipitation solution, the acetate can be one of sodium acetate and potassium acetate, and the concentration of acetate is 3 to 3.6 M; the concentration of acetic acid is 2 to 3.2 M.

[0020] In the Buffer 3 binding solution, the concentration of guanidine hydrochloride is 1 to 2.5 M, and the Buffer 3 binding solution is prepared with a mixture of Tris-Hcl and EDTA, wherein the volume ratio of Tris-Hcl to EDTA is (10 to 15):(1 to 2).

[0021] The supernatant after sample lysis and precipitation needs to be transferred to the adsorption plate and 1.5 times the binding solution is added; after sample lysis and precipitation, it can be placed at room temperature or 65°C for 10 minutes and centrifuged at maximum centrifugal force for 5 to 10 minutes; the rinsing solution requires an ethanol concentration between 70% and 85%; the elution solution has a pH between 7 and 8.

[0022] Plants include potato, barley, wheat, and Arabidopsis.

[0023] Example 1: Using potatoes as samples, high-quality genomic DNA was extracted.

[0024] The extraction reagent includes the following components in mass fraction or volume fraction:

[0025] Buffer 1 lysis buffer: 500mM sodium chloride, 100mM Tris-HCl (pH 7.4), 1% PVP40, 100mM sodium metabisulfite, 2% SDS, 10% isopropanol, 50mM EDTA;

[0026] Buffer 2 precipitation solution: 3.6 M potassium acetate, 2.4 M acetic acid;

[0027] Buffer 3 binding solution: 2M guanidine hydrochloride (prepare 1000ml, add 191g guanidine hydrochloride, make up to 333ml with TE (Tris EDTA 10:1, pH 8), add 667ml anhydrous ethanol);

[0028] Buffer 4 rinse solution: To prepare 1000 ml, add 2 ml 1M Tris-Hcl (pH 8), 800 ml anhydrous ethanol, and make up the rest to 1000 ml with ddH2O;

[0029] Buffer 5: 10mM Tris-HCl (pH8), 1mM EDTA

[0030] Cell lysis: Place 50 mg of fresh tissue and two 5 / 32 inch (3.97 mm) stainless steel beads in a 1ml deep-well 96-well plate in each well. 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, grind the tissue at 30Hz for 1 minute, and then repeat the above operation after changing the orientation of the plate in the adapter for more uniform grinding. If only one plate is homogenized 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 the well. Mix the seal thoroughly by shaking it up and down manually (do not use a vortexer).

[0031] 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 room temperature at maximum centrifugal force for 10 minutes. 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 1 ml filter plate and place 1.1ml 96-well deep-well plate, centrifuge at 4000×g for 2 minutes at room temperature. Pour off the flow-through and wipe the surface of the collection plate with a paper towel, wash each well with 300μl Buffer 4 rinse solution. 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 new one. 1.1ml 96-well deep-well plate, used to collect DNA to be eluted from the filter plate. Add 50μl of Buffer 5 to each well, let stand for 1 minute, and then centrifuge at 4000×g for 1 minute at room temperature to obtain the genomic extract.

[0032] The results of the measurement of the genomic extract using the Thermo fishernanodrop are shown in Table 1.

[0033] Example 2: Using barley leaves as samples, high-quality genomic DNA was extracted.

[0034] The extraction reagent includes the following components in mass fraction or volume fraction:

[0035] Buffer 1 lysis buffer: 500 mM NaCl, 100 mM Tris-HCl (pH 7.4), 1% PVP40, 2% SDS, 10% isopropanol, 50 mM EDTA;

[0036] Buffer 2 precipitation solution: 3.6 M potassium acetate, 2.4 M acetic acid;

[0037] Buffer 3 binding solution: 2M guanidine hydrochloride (prepare 1000ml, add 191g guanidine hydrochloride, make up to 333ml with TE (Tris EDTA 10:1, pH 8), add 667ml anhydrous ethanol);

[0038] Buffer 4 rinse solution: To prepare 1000 ml, add 2 ml 1M Tris-Hcl (pH 8), 800 ml anhydrous ethanol, and make up the rest to 1000 ml with ddH2O;

[0039] Buffer 5: 10 mM Tris-HCl (pH 8), 1 mM EDTA;

[0040] Cell lysis: Place 50 mg of fresh tissue and two 5 / 32 inch (3.97 mm) stainless steel beads in a 1ml deep-well 96-well plate in each well. 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, grind the tissue at 30Hz for 1 minute, and then repeat the above operation after changing the orientation of the plate in the adapter for more uniform grinding. If only one plate is homogenized 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 the well. Mix the seal thoroughly by shaking it up and down manually (do not use a vortexer).

[0041] 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 room temperature at maximum centrifugal force for 10 minutes. 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 1 ml filter plate and place 1.1ml 96-well deep-well plate, centrifuge at 4000×g for 2 minutes at room temperature. Pour off the flow-through and wipe the surface of the collection plate with a paper towel, wash each well with 300μl Buffer 4 rinse solution. 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 new one. 1.1ml 96-well deep-well plate, used to collect DNA to be eluted from the filter plate. Add 50μl of Buffer 5 to each well, let stand for 1 minute, and then centrifuge at 4000×g for 1 minute at room temperature to obtain the genomic extract.

[0042] The results of the measurement of the genomic extract using the Thermo fishernanodrop are shown in Table 1.

[0043] Example 3: Using wheat leaves as samples, high-quality genomic DNA was extracted.

[0044] The extraction reagent includes the following components in mass fraction or volume fraction:

[0045] Buffer 1 lysis buffer: 500mM sodium chloride, 100mM Tris-HCl (pH 7.4), 1% PVP40, 100mM sodium metabisulfite, 2% SDS, 10% isopropanol, 50mM EDTA;

[0046] Buffer 2 precipitation solution: 3.6 M sodium acetate, 2.4 M acetic acid;

[0047] Buffer 3 binding solution: 2M guanidine hydrochloride (prepare 1000ml, add 191g guanidine hydrochloride, make up to 333ml with TE (Tris EDTA 10:1, pH 8), add 667ml anhydrous ethanol);

[0048] Buffer 4 rinse solution: To prepare 1000 ml, add 2 ml 1M Tris-Hcl (pH 8), 800 ml anhydrous ethanol, and make up the rest to 1000 ml with ddH2O;

[0049] Buffer 5: 10 mM Tris-HCl (pH 8), 1 mM EDTA;

[0050] Cell lysis: Place 50 mg of fresh tissue and two 5 / 32 inch (3.97 mm) stainless steel beads in a 1ml deep-well 96-well plate in each well. 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, grind the tissue at 30Hz for 1 minute, and then repeat the above operation after changing the orientation of the plate in the adapter for more uniform grinding. If only one plate is homogenized 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 the well. Mix the seal thoroughly by shaking it up and down manually (do not use a vortexer).

[0051] 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 room temperature at maximum centrifugal force for 10 minutes. 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 1 ml filter plate and place 1.1ml 96-well deep-well plate, centrifuge at 4000×g for 2 minutes at room temperature. Pour off the flow-through and wipe the surface of the collection plate with a paper towel, wash each well with 300μl Buffer 4 rinse solution. 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 new one. 1.1ml 96-well deep-well plate, used to collect DNA to be eluted from the filter plate. Add 50μl of Buffer 5 to each well, let stand for 1 minute, and then centrifuge at 4000×g for 1 minute at room temperature to obtain the genomic extract.

[0052] The results of the measurement of the genomic extract using the Thermo fishernanodrop are shown in Table 1.

[0053] Example 4: Extracting high-quality genomic DNA from tomato leaves.

[0054] The extraction reagent includes the following components in mass fraction or volume fraction:

[0055] Buffer 1 lysis buffer: 1000mM sodium chloride, 100mM Tris-HCl (pH 7.4), 1% PVP40, 100mM sodium metabisulfite, 2% SDS, 10% isopropanol, 50mM EDTA;

[0056] Buffer 2 precipitation solution: 3.6 M potassium acetate, 2.4 M acetic acid;

[0057] Buffer 3 binding solution: 2M guanidine hydrochloride (prepare 1000ml, add 191g guanidine hydrochloride, make up to 333ml with TE (Tris EDTA 10:1, pH 8), add 667ml anhydrous ethanol);

[0058] Buffer 4 rinse solution: To prepare 1000 ml, add 2 ml 1M Tris-Hcl (pH 8), 800 ml anhydrous ethanol, and make up the rest to 1000 ml with ddH2O;

[0059] Buffer 5: 10 mM Tris-HCl (pH 8), 1 mM EDTA;

[0060] Cell lysis: Place 50 mg of fresh tissue and two 5 / 32 inch (3.97 mm) stainless steel beads in a 1ml deep-well 96-well plate in each well. 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, grind the tissue at 30Hz for 1 minute, and then repeat the above operation after changing the orientation of the plate in the adapter for more uniform grinding. If only one plate is homogenized 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 the well. Mix the seal thoroughly by shaking it up and down manually (do not use a vortexer).

[0061] 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 room temperature at maximum centrifugal force for 10 minutes. 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 1 ml filter plate and place 1.1ml 96-well deep-well plate, centrifuge at 4000×g for 2 minutes at room temperature. Pour off the flow-through and wipe the surface of the collection plate with a paper towel, wash each well with 300μl Buffer 4 rinse solution. 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 new one. 1.1ml 96-well deep-well plate, used to collect DNA to be eluted from the filter plate. Add 50μl of Buffer 5 to each well, let stand for 1 minute, and then centrifuge at 4000×g for 1 minute at room temperature to obtain the genomic extract.

[0062] The results of the measurement of the genomic extract using the Thermo fishernanodrop are shown in Table 1.

[0063] Example 5: Using Arabidopsis thaliana as a sample, high-quality genomic DNA was extracted.

[0064] The extraction reagent includes the following components in mass fraction or volume fraction:

[0065] Buffer 1 lysis buffer: 500mM sodium chloride, 100mM Tris-HCl (pH 7.4), 1% PVP40, 100mM sodium metabisulfite, 2% SDS, 10% isopropanol, 50mM EDTA;

[0066] Buffer 2 precipitation solution: 3.6 M potassium acetate, 2.4 M acetic acid;

[0067] Buffer 3 binding solution: 1M guanidine hydrochloride (prepare 1000ml, add 95.5g guanidine hydrochloride, make up to 333ml with TE (Tris EDTA 10:1, pH 8), add 667ml anhydrous ethanol);

[0068] Buffer 4 rinse solution: To prepare 1000 ml, add 2 ml 1M Tris-Hcl (pH 8), 800 ml anhydrous ethanol, and make up the rest to 1000 ml with ddH2O;

[0069] Buffer 5: 10 mM Tris-HCl (pH 8), 1 mM EDTA;

[0070] Cell lysis: Place 50 mg of fresh tissue and two 5 / 32 inch (3.97 mm) stainless steel beads in a 1ml deep-well 96-well plate in each well. 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, grind the tissue at 30Hz for 1 minute, and then repeat the above operation after changing the orientation of the plate in the adapter for more uniform grinding. If only one plate is homogenized 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 buffer and 6ul RNase (10mg / ml) from the reservoir to each well using a multichannel pipette and cover the well. Mix the seal thoroughly by shaking it up and down manually (do not use a vortexer).

[0071] 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 room temperature at maximum centrifugal force for 10 minutes. 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 1 ml filter plate and place 1.1ml 96-well deep-well plate, centrifuge at 4000×g for 2 minutes at room temperature. Pour off the flow-through and wipe the surface of the collection plate with a paper towel, wash each well with 300μl Buffer 4 rinse solution. 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 new one. 1.1ml 96-well deep-well plate, used to collect DNA to be eluted from the filter plate. Add 50μl of Buffer 5 to each well, let stand for 1 minute, and then centrifuge at 4000×g for 1 minute at room temperature to obtain the genomic extract.

[0072] The results of the measurement of the genomic extract using the Thermo fishernanodrop are shown in Table 1.

[0073] Example 6: The difference between this example and Example 1 is the extraction and purification of nucleic acid: 130ul of Buffer 2 precipitation solution was added to the above treated solution, and the The sealing pad was placed at room temperature for 10 minutes and then placed in a 65°C water bath for 10 minutes. The genome extract was measured using a Thermo fishernanodrop, and the results are shown in Table 1.

[0074] Example 7: The difference between this example and Example 1 is that the 2% SDS in Buffer 1 lysis solution was changed to 1% SDS, and the results of the genome extraction solution measurement by Thermo fishernanodrop are shown in Table 1.

[0075] Example 8: The difference between this example and Example 1 is that the lysis buffer in Buffer 1 was changed to 50 mM sodium metabisulfite, and the results of the genomic extract measurement using Thermo fishernanodrop are shown in Table 1.

[0076] Example 9: The difference between this example and Example 1 is that the lysis buffer 1 was changed to 0.5% PVP40, and the genome extract was measured by Thermo fishernanodrop. The results are shown in Table 1.

[0077] Table 1

[0078]

[0079]

[0080] Note: When measured using a Thermo fishernanodrop UV spectrophotometer, the OD260 / OD280 ratio is generally between 1.7-1.9, and the OD260 / OD230 is between 2.0-2.5. If buffer 5 is not used for elution but ddH2O is used, the ratio will be lower because pH value and the presence of ions will affect the absorbance, but it does not mean that the purity is low.

[0081] It can be seen from the results of Table 1 that the lysate, precipitate and binding solution used in the present invention have high quality of genomic DNA extracted; Examples 1 and 2 show that the dual components of PVP40 and sodium metabisulfite can improve the removal rate of polyphenols, but neither of them can do it alone; Examples 1 and 3 show that potassium acetate is more capable of precipitating and removing polysaccharides in 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 Buffer 2 precipitate is added to the above-treated solution, and the solution is covered with a precipitate. The genome yield of the sealing pad was higher when it was placed in a 65°C water bath for 10 minutes instead of at room temperature; Examples 1 and 7 showed that 2% SDS had a better lysis effect than 1% SDS. It can be seen that Example 6 had the best effect and the extracted genome concentration was the highest.

[0082] 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).

[0083] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. Plant genomic DNA extraction reagent, characterized by: Includes 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; wherein the concentration of acetate is 2.2 to 4.3 M; 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 solution comprises sodium chloride, Tris-Hcl, PVP40, sodium metabisulfite, SDS and isopropanol; the concentration of the sodium chloride is 500-1000mM; the concentration of the Tris-Hcl is 50-100mM; the mass percentage of PVP40 is 1-2%; the concentration of the sodium metabisulfite is 50-100mM; the mass percentage of the SDS is 1-2%; and the volume percentage of the isopropanol is 5-10%.

6. The plant genomic DNA extraction reagent according to any one of claims 1 to 5, characterized in that: In the Buffer 2 precipitation solution, the acetate is potassium acetate or sodium acetate.

7. The plant genomic DNA extraction reagent according to claim 6, characterized in that: In the Buffer 2 precipitation solution, the concentration of acetate is 3-3.6M.

8. The plant genomic DNA extraction reagent according to claim 7, characterized in that: In the Buffer 2 precipitation solution, the Buffer 2 precipitation solution includes acetate and acetic acid; the concentration of acetate is 3 to 3.6M; the concentration of acetic acid is 2 to 3.2M.

9. The plant genomic DNA extraction reagent according to claim 7 or 8, characterized in that: In the Buffer 3 binding solution, the concentration of guanidine hydrochloride is 1-2.5M.

10. The plant genomic DNA extraction reagent according to claim 9, characterized in that: The Buffer 3 binding solution is prepared with 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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