CN112029762A - Plant tissue DNA rapid extraction method, extraction kit and extraction device - Google Patents
Plant tissue DNA rapid extraction method, extraction kit and extraction device Download PDFInfo
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- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- B02C19/00—Other disintegrating devices or methods
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Abstract
A method for extracting plant tissue DNA comprises adding plant tissue into a grinding device; pre-cooling the plant tissue and a grinding device, wherein the moisture of the plant tissue is condensed after the pre-cooling, and the grinding device reaches a temperature after the pre-cooling, which enables the moisture to keep a condensed state when the plant tissue is ground therein; grinding the plant tissue by using a pre-cooled grinding device under the condition of no protection of a liquid refrigerant to obtain plant tissue powder; performing DNA extraction on the plant tissue powder. And an extraction kit and an extraction apparatus for carrying out the method. The method for extracting the DNA of the plant tissue can greatly shorten the operation time, simplify the operation steps, improve the experimental efficiency, does not use toxic reagents, is beneficial to the health of operators and reduces the damage to the environment.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the technical field of bioengineering, and particularly relates to a method, a kit and a device for rapidly extracting plant tissue DNA.
[ background of the invention ]
With the development of genome sequencing technology, the research on the sequence, structure and function of plant genome is rapidly developed, and the acquisition of plant genome DNA with high purity, high content and high integrity is the first prerequisite for the application of genome sequencing technology. The extraction of plant genome DNA is more difficult than the extraction of microorganism, animal tissue or blood sample genome because of the large amount of polysaccharide, polyphenol substances and other secondary metabolites with complex structures in the plant tissue.
The traditional plant genome DNA extraction method mainly comprises a CTAB method and an SDS method, and the extraction steps mainly comprise the steps of grinding plant tissues under the protection of liquid nitrogen, and then the processes of cell lysis, impurity removal, DNA precipitation, rinsing, elution and the like. It mainly uses detergents such as CTAB or SDS to crack cells and releases genome DNA in the cells; then removing impurities such as protein, polyphenol, polysaccharide and the like by a method of chloroform and phenol extraction or high-salt precipitation; then adding a proper volume of organic solvent such as absolute ethyl alcohol, isopropanol or PEG and the like into the extracted supernatant to precipitate or adsorb the DNA on media such as a silica gel column, an ion exchange column and the like; finally, after rinsing with a rinsing solution, the solution is dissolved by a low-concentration salt solution or eluted from the medium.
Patent document CN 110592072a discloses a method for extracting plant genome DNA and its application, which comprises grinding plant tissue under the protection of liquid nitrogen, adding plant tissue powder into CTAB lysate at 65 ℃ and incubating for 40 minutes, then extracting DNA, the total operation time is more than 2.5 hours, and toxic substances such as beta-mercaptoethanol are needed.
Patent document CN 110564722a discloses a method for extracting genomic DNA of paphiopedilum by using an improved CTAB method, which comprises crushing leaves at ultra-low temperature, adding CTAB lysate and performing ultrasonic assisted incubation at 65 ℃, phenol, chloroform resonance emulsification and the like, wherein the total operation time is not less than 1.5 hours, toxic substances such as beta-mercaptoethanol, phenol, chloroform and the like are required, and special instruments such as ultrasound and the like are required.
Patent document CN 110358762a discloses a method for extracting plant leaf genome DNA, which comprises placing plant leaves into a multichannel homogenizer for disruption, incubating for 45 minutes at 65 ℃ in a lysis solution, and extracting DNA by a magnetic bead method, wherein the total operation time is not less than 1 hour, toxic substances such as beta-mercaptoethanol and guanidine hydrochloride are required, and the requirements for instruments are complex.
Patent document CN 109439652a discloses a method for extracting polysaccharide-rich plant genome DNA, which comprises grinding plant tissues under the protection of liquid nitrogen, lysing plant cells with CTAB, and further performing DNA precipitation extraction with phenol, chloroform and isopropanol, wherein the total operation time is not less than 1 hour, and toxic substances such as β -mercaptoethanol, phenol, chloroform and the like are required.
Patent document CN109136221A discloses a method for extracting total DNA from rubber tree leaves, which comprises grinding plant leaves into powder in liquid nitrogen, incubating in CTAB lysate at 65 ℃ for 40 minutes, and extracting DNA by phenol, chloroform and isopropanol, wherein the total operation time is not less than 3.5 hours, and toxic substances such as beta-mercaptoethanol, phenol, chloroform and the like are required.
As can be seen from the above patent documents, the existing methods for extracting DNA from plant tissues generally have the problems of long operation time (generally longer than 1 hour or even 2 hours), use of toxic substances (phenol, chloroform, β -mercaptoethanol), and the like, and the long operation time causes degradation of DNA, wastes precious time of experimenters, and reduces experimental efficiency; the use of toxic substances can cause harm to physical and mental health of operators, the waste discharge has adverse effect on the environment, the post-treatment cost is high, and the requirements of sustainable development are not met.
[ summary of the invention ]
The present invention is directed to overcoming the above-mentioned disadvantages of the prior art and providing a method for rapidly extracting DNA from plant tissues, and a kit and an extraction apparatus for performing the method.
In order to achieve the above object, the present invention provides in a first aspect a method for extracting plant tissue DNA, comprising the steps of:
(a) adding plant tissue to a grinding device or grinder;
(b) pre-cooling the plant tissue and a grinding device, wherein the moisture of the plant tissue is condensed after the pre-cooling, and the grinding device reaches a temperature after the pre-cooling, which enables the moisture to keep a condensed state when the plant tissue is ground therein;
(c) grinding the plant tissue by using a pre-cooled grinding device under the condition of no protection of a liquid refrigerant to obtain plant tissue powder;
(d) performing DNA extraction on the plant tissue powder.
The plants include, but are not limited to, aquatic plants, terrestrial plants, wherein the aquatic plants include floating plants and submerged plants, wherein the terrestrial plants include scindapsus aureus, rosa chinensis, camellia japonica, buxus microphylla, photinia fraseri, rosa chinensis, lactuca indica, buxus macrocarpa, willow, asparagus lettuce, machilus humilis, japanese coral tree, soybean, and the like; the plant tissue comprises one of roots, stems, leaves, flowers and fruits of the plant or any combination thereof.
The grinding device is selected from plant tissue disruption devices commonly used in the art, typically from a mortar or a grinder, but other devices capable of achieving the same purpose may be employed. In the case of polyphenol polysaccharide plant tissues which are difficult to grind in grinding, 50 mesh quartz sand is preferably added in order to sufficiently crush the plant tissues, and the quartz sand may be removed in a subsequent centrifugation step.
Preferably, the pre-cooling in step (b) is performed by adding a liquid refrigerant to the grinding device loaded with the plant tissue; the addition amount of the liquid refrigerant can enable the moisture of the plant tissue to be condensed after precooling, the grinding device to reach the temperature capable of keeping the moisture in a condensed state when the plant tissue is ground in the grinding device after precooling, and the liquid refrigerant volatilizes into a gaseous state to be dissipated after precooling the plant tissue and the grinding device.
The liquid cryogen is typically selected from liquid nitrogen, which is also required for "grinding under liquid nitrogen protection" as is generally taught in the art. In fact, any liquid cryogen that achieves a similar purpose may be used, such as liquid helium. However, liquid nitrogen is more preferable from the viewpoint of availability and price of the reagent.
As a preferred option, the grinding of step (c) is completed within 10 minutes, preferably within 5 minutes.
As a preferred embodiment, step (d) further comprises:
i. transferring the plant tissue powder into a container (typically an EP tube) containing the first solution and mixing (preferably, mixing by vortex shaking for 10-30 seconds);
adding the second solution and mixing (preferably, mixing gently and rapidly for 10-30 seconds), centrifuging (preferably, at 12000rpm for about 13400g for 1-2 minutes) and retaining the supernatant (preferably, transferring the supernatant into a new centrifuge tube);
adding the third solution, uniformly mixing, transferring into an adsorption column, centrifuging (preferably at the rotation speed of 12000rpm, about 13400g, centrifuging for 20-40 seconds) and discarding the filtrate;
adding the fourth solution to the adsorption column, centrifuging (preferably at 12000rpm, about 13400g, centrifuging for 20-40 seconds) and discarding the filtrate;
v. fully drying the adsorption column (preferably, standing for 1-3 minutes at room temperature), and transferring to a new container;
and vi, dropwise adding the fifth solution into the center of the adsorption column, standing at room temperature (preferably for 1-3 minutes), centrifuging (preferably at 12000rpm at about 13400g for 30 seconds-2 minutes), and collecting filtrate to obtain the plant tissue DNA.
The adsorption column is selected from DNA adsorption columns commonly used in nucleic acid extraction in the field, typically a silica membrane adsorption column, which has strong adsorption effect on DNA under acidic conditions, and has significantly reduced adsorption effect on DNA under alkaline conditions, and is commonly used in DNA extraction.
As a preferred scheme, the steps i to vi are all completed in a room temperature environment; to increase DNA purity, step iv can be repeated several times, typically 1, 2 or 3 times; in order to increase the DNA yield, step vi can be repeated 1 time, i.e., the filtrate is added to the same adsorption column again and centrifuged.
As a preferred embodiment, the first solution is a lysate comprising CTAB, Tris-HCl, EDTA, glycerol and NaCl, 10mg/ml RNase (added when extracting DNA); the concentration of CTAB is 2-5%, the concentration of Tris-HCl is 50-100 mM, the concentration of EDTA is 10-20 mM, the concentration of glycerol is 1-4%, the concentration of NaCl is 2.0-2.4M, and the concentration of 10mg/ml RNase (added during DNA extraction) is 4-20 ul.
As a preferable scheme, the second solution is a NaCl solution with the concentration of 4-5M; the third solution is isopropanol, and the ratio of the addition amount of the isopropanol to the volume of the supernatant obtained in the step ii is 0.4-0.8: 1; the fourth solution is rinsing liquid containing 65-80% of ethanol; the fifth solution is generally TE eluent, the pH value of which is in the range of 7.0-8.5, and if the pH value of deionized water obtained by a water making machine is in the range, pure water can be directly used for elution.
By the operation, the DNA of the plant tissue can be extracted within 20 minutes, even 15 minutes.
The present invention provides in a further aspect a kit for carrying out the aforementioned plant tissue DNA method, said kit comprising: a first solution containing CTAB with a concentration of 2-5%, Tris-HCl with a concentration of 50-100 mM, EDTA with a concentration of 10-20 mM, 1-4% glycerol, 4-20 ul RNase with a concentration of 10mg/ml (added when extracting DNA) and 2.0-2.4M NaCl; and a vector for describing the DNA method for plant tissues.
The vector describing the DNA method of the plant tissue is typically a paper specification, but may be any vector describing an electronic version of the method (including but not limited to a removable disk, an optical disk, an electronic ink screen, a network resource, an address thereof, etc.), and is within the scope of the present concept as long as the method can be known by reading the vector.
In another aspect, the present invention provides a computer readable medium carrying a computer program comprising instructions for carrying out the method for DNA-based expression of plant tissue as described above. The computer is understood in a broad sense and includes but is not limited to a single chip microcomputer, a PLC, a single chip microcomputer, an industrial personal computer, a PC and the like. The computer readable carrier includes, but is not limited to, any form of Flash, EEPROM, magnetic disk (floppy or hard disk), optical disk, and the like. The computer program may be written in any language, such as assembly, JAVA, VB, VC, C + +, Python, as long as the associated system is controlled to implement the method.
The present invention provides in another aspect an automated nucleic acid extraction apparatus comprising: a liquid filling/pipetting assembly, a centrifugation assembly, a grinding assembly and a controller electrically connected with the assemblies; the controller comprises the aforementioned computer readable carrier. Wherein the liquid filling/pipetting assembly is used for controlling the filling and removing of various liquids in the aforementioned method, the centrifugation assembly and the grinding assembly are respectively used for performing corresponding centrifugation and grinding operations, and the controller is used for controlling the aforementioned assemblies to automatically perform the aforementioned plant tissue DNA extraction method. An automated nucleic acid isolation apparatus is widely known in the art, for example, see patent document CN 110305767A, CN 1431290A, CN 1158951a, etc., and thus is not described herein in detail.
The inventor surprisingly found in the research of the plant tissue DNA extraction method that when the plant tissue is cryogenically ground, the removal of the liquid nitrogen protection can obtain plant tissue powder with more sufficient fragmentation in a shorter time, which is more beneficial for the subsequent extraction operation, for example, the CTAB incubation step can be omitted, the experimental process is greatly shortened, and the quality of DNA extraction is ensured (further proved in the specific implementation mode).
Unlike the grinding under protection of liquid nitrogen generally taught by the prior art, the inventors precool the plant tissue and the grinding device under a small amount of liquid nitrogen, the plant tissue is frozen to show brittleness in a relatively short time, and meanwhile, the grinding device is precooled to a state of being kept at a lower temperature (for example, lower than zero degree) for a period of time, and the liquid nitrogen is volatilized into the atmosphere; thus, the brittle plant tissue does not float around in the mortar due to the presence of liquid nitrogen, and thus finer plant tissue powder can be obtained in a shorter time. When the plant tissue powder is finer than the conventional one, the contact area with the lysis solution is larger (for example, more than 1 order of magnitude difference), so that the cell lysis and the DNA release can be completed in a shorter time. Since the grinding device and the plant tissue are always in a state of being lower than zero (or lower) in the grinding, the DNA in the plant tissue is not degraded in this period, which ensures the quality of extraction.
The plant tissue DNA extraction method, the extraction kit and the extraction device provided by the invention at least have the following beneficial effects:
1. the plant after the instant cooling is ground under the drying condition without the protection of liquid nitrogen, so that the operation time is greatly shortened, the operation steps are simplified, and the experiment efficiency is improved;
2. toxic reagents are not used, so that the health of operators is facilitated, and the damage to the environment is reduced;
3. and conventional reagents and instruments are adopted, so that the operability is stronger and the cost is lower.
And 4, the DNA extraction process is green and environment-friendly, and is beneficial to the health of experimenters and reduces the damage to the environment.
[ description of the drawings ]
FIG. 1 is a graph of temperature rise after precooling of the grinding system;
FIG. 2 is a gel electrophoresis chart of CTAB extraction results of different concentrations in the method of the invention;
FIG. 3 is a gel electrophoresis image of 4 plant tissue DNAs extracted by the method of the present invention;
FIG. 4 is a gel electrophoresis image of the DNA extracted from 7 plant tissues by the method of the present invention.
FIG. 5 is a gel electrophoresis image of DNA extracted from old leaves, roots, stems, seeds, sweet potato pulp and apples by the method of the invention.
FIG. 6 is a gel electrophoresis image of the method of the present invention for extracting 7 plant tissue DNAs by grinding with an intelligent sample grinder.
FIG. 7 is an electrophoretogram showing the results of DNA extraction by the method of the present invention using 5M PH7.0 NaAc instead of 5M NaCl and 5M PH7.2 KAc and using 5M PH7.0KAc instead of 5M NaCl scindapsus aureus.
FIG. 8 is an electrophoretogram of the results of DNA extraction from scindapsus aureus leaves at pH4.0 NaAc instead of pH8.0 Tris-HCl according to the method of the invention.
[ detailed description ] embodiments
The invention is further described below in conjunction with the drawings and the specific embodiments, which are provided only to assist in understanding the invention.
EXAMPLE 1 feasibility of grinding temperature and time
As mentioned above, the particularity of the present invention resides primarily in the manner of polishing without liquid nitrogen protection. To ensure that plant tissue DNA does not degrade during milling in this manner, the inventors examined the time-temperature curve of ambient heat exchange with a mortar after liquid nitrogen pre-cooling using a mortar common in the art (fig. 1). Immediately after a small amount of liquid nitrogen had pre-cooled and evaporated, the mortar reached a temperature below-50 ℃ (temperature in this range was not detectable by the thermometric instruments used by the inventors). Thereafter, the temperature of the milling system increased as the heat exchange between the environment and the mortar proceeded, but was below-35 ℃ in 5 minutes and still below-20 ℃ in 10 minutes. -20 ℃ is a common DNA storage temperature in the art, at which DNA is not susceptible to degradation, i.e. grinding of plant tissue within 10 minutes does not cause DNA degradation even without liquid nitrogen protection. Under the condition of no liquid nitrogen, the plant tissues can not randomly move in the mortar due to the boiling of the liquid nitrogen, the grinding efficiency is greatly improved, the fineness equivalent to 10 minutes of grinding under the protection of the liquid nitrogen can be achieved within 2 minutes, and the particle fineness required by extracting the DNA of the plant tissues can be achieved within 5 minutes.
Example 2 determination of the optimal concentration of Components in the lysate
The scindapsus aureus leaves were subjected to DNA extraction according to the method described above, ground for 5 minutes, and the optimal concentration range of CTAB was determined by one-way analysis. Weighing scindapsus aureus leaf at 390 mg. Wherein the first solution (buffered lysate) comprises Tris-HCl 100mM, EDTA 20mM, glycerol 3%, NaCl 2.0M, 8ul 10mg/ml RNase (added during DNA extraction), and CTAB at 2%, 3%, 4%, 5%, 6% by volume concentration, respectively. The results were determined on a biaosharp (table 1) and examined by gel electrophoresis (fig. 2), showing that 2% to 5% CTAB gave better results, 3% being optimal, and the plant genomic DNA fragments were all more intact and less degraded fragments.
Table 1: quality of plant tissue DNA extracted with different CTAB concentrations
| |
2％ | 3％ | 4％ | 5％ | 6％ |
| DNA concentration | 63.92ng/ul | 72.60ng/ul | 69.95ng/ul | 64.91ng/ul | 54.81ng/ul |
| A260/A280 | 1.842 | 1.837 | 1.817 | 1.826 | 1.843 |
The optimum concentration range of NaCl was determined by one-way analysis and ground for 5 minutes. Wherein the first solution contains Tris-HCl 100mM, EDTA 20mM, glycerol 3%, CTAB 3%, 8ul 10mg/ml RNase (added when extracting DNA), and NaCl with the concentration of 2.0, 2.2, 2.4, 2.6, 2.8, 3.0M respectively, and the amount of the scindapsus aureus leaf is about 543 mg. The results were measured on a biaosharp (Table 2), and it was found that NaCl was preferable at 2.0 to 2.4M, and 2.4M was most preferable.
Table 2: quality of DNA extracted from plant tissue at different NaCl concentrations
| Concentration of NaCl | 2.0M | 2.2M | 2.4M | 2.6M | 2.8M | 3.0M |
| DNA concentration | 54.86ng/ul | 61.89ng/ul | 64.07ng/ul | 35.87ng/ul | 35.36ng/ul | 13.18ng/ul |
| A260/A280 | 1.846 | 1.851 | 1.845 | 1.809 | 1.814 | 1.809 |
The optimal concentration range of glycerol was determined by one-way analysis and milled for 5 minutes. Wherein the first solution comprises Tris-HCl 100mM, EDTA 20mM, NaCl 2.4M, CTAB 3%, 8ul 10mg/ml RNase (added during DNA extraction), and glycerol with volume concentration of 1%, 2%, 3%, 4%, and about 363mg of scindapsus aureus leaf is weighed. The results were measured on a biaosharp (table 3), and it was found that 1% to 4% of glycerol gave better results, and 3% was the best.
Table 3: quality of plant tissue DNA extracted at different glycerol concentrations
The optimum concentration range of EDTA was determined by one-way analysis and milling for 5 minutes. Wherein the first solution comprises Tris-HCl 100mM, NaCl 2.4M, CTAB 3%, glycerol 3%, 8ul 10mg/ml RNase (added during DNA extraction), and EDTA with concentration of 10, 20, 30, 40mM respectively, and the amount of the leaf of scindapsus aureus is about 372 mg. As a result of measurement on biaosharp (Table 4), it was found that EDTA was preferably present at 10 to 20mM, and most preferably at 20 mM.
Table 4: quality of plant tissue DNA extracted at different EDTA concentrations
| EDTA concentration | 10mM | 20mM | 30mM | 40mM |
| DNA concentration | 60.14ng/ul | 70.05ng/ul | 53.68ng/ul | 33.68ng/ul |
| A260/A280 | 1.801 | 1.817 | 1.805 | 1.807 |
The optimal concentration range of Tris-HCl was determined by one-way assay and ground for 5 minutes. Wherein the first solution contains EDTA 20mM, NaCl 2.4M, CTAB 3%, glycerol 3%, 8ul 10mg/ml RNase (added during DNA extraction), and EDTA with concentration of 50, 100, 150, 200mM respectively, and the amount of the leaf of scindapsus aureus is about 399 mg. As a result of measurement on biaosharp (Table 5), 50 to 100mM Tris-HCl was preferable, and 100mM was most preferable.
| Tris-HCl gradient | 50mM | 100mM | 150mM | 200mM |
| Concentration of | 44.88ng/ul | 59.33ng/ul | 36.64 | 35.73 |
| A260/A280 | 1.804 | 1.835 | 1.775 | 1.811 |
Example 3 optimal volume ratio of Isopropanol
The scindapsus aureus leaves were subjected to DNA extraction according to the method described above, ground for 5 minutes and the optimal volume ratio of isopropanol to centrifuged supernatant in step iii was determined by one-way analysis. The amount of the scindapsus aureus leaf was measured to be about 371 mg. The results were measured on a biaosharp (table 5), and it was found that 0.5 to 0.8 times by volume of isopropanol gave better results, with 0.7 times by volume being the best.
Table 5: quality of plant tissue DNA extracted from different isopropanol volume ratios
| Volume of isopropyl alcohol | 0.5V | 0.6V | 0.7V | 0.8V |
| DNA concentration | 74.73ng/ul | 87.14ng/ul | 93.41ng/ul | 91.72ng/ul |
| A260/A280 | 1.807 | 1.813 | 1.822 | 1.819 |
Example 4 DNA extraction of different plant tissues
According to the optimal experimental conditions determined above, various plant tissues are extracted according to the following methods.
(1) Weighing about 100mg of plant tissues and adding the plant tissues into a grinding device;
(2) adding a small amount of liquid nitrogen into a grinding device for precooling;
(3) continuously grinding the plant tissue for 5 minutes after the liquid nitrogen is volatilized to obtain plant tissue powder;
(4) transferring the plant tissue powder into an EP tube containing lysis solution and RNA enzyme, and mixing uniformly for 20 seconds by vortex oscillation;
(5) add 75. mu.l of 5M NaCl solution and mix gently and quickly for 20 seconds, centrifuge at 12000rpm (about 13400g) for 1 minute and transfer the supernatant to a new EP tube;
(6) adding isopropanol with the volume of 0.4-0.7 times of that of the mixture, uniformly mixing, transferring the mixture into a silicon dioxide membrane adsorption column, centrifuging the mixture for 30 seconds at the rotating speed of 12000rpm (about 13400g), and removing the filtrate;
(7) adding a rinsing solution containing 70% ethanol to the adsorption column, centrifuging at 12000rpm (about 13400g) for 30 seconds, sucking away the filtrate, and rinsing repeatedly once;
(8) the column was left at room temperature for 2 minutes to dry thoroughly and transferred to a new EP tube;
(9) TE eluent is dripped into the center of the adsorption column, the adsorption column is placed at room temperature for 2 minutes and then centrifuged, the rotation speed is 12000rpm (about 13400g) is centrifuged for 1 minute, filtrate is collected, and plant tissue DNA is obtained and is respectively subjected to bioaosharp and gel electrophoresis analysis to obtain a table 6, a table 7, a table 8, a figure 3, a figure 4 and a figure 5.
Table 6: extraction of DNA quality of 4 plant tissues
| Plant tissue name | Camellia petal | Ramulus Et folium Buxi Sinicae | Photinia fraseri | Chinese redbud dwarf cherry |
| Quality of | 98.2mg | 98.8mg | 100.2mg | 97.1mg |
| DNA concentration | 18.71ng/ul | 113.7ng/ul | 146.2ng/ul | 129.6ng/ul |
| A260/A280 | 1.812 | 1.807 | 1.813 | 1.836 |
Table 7: extraction of DNA quality of 7 plant tissues
Table 8: extraction of DNA quality of 6 plant tissues
| Plant tissue name | Lao Lu Ye | Old potato stem | Wheat seed | Sweet potato meat | Old root of potato | Apple meat |
| Quality of | 98.1mg | 100.1mg | 142.7mg | 100.0mg | 99.8mg | 110.2mg |
| DNA concentration | 33.94ng/ul | 23.70ng/ul | 75.32ng/ul | 32.13ng/ul | 42.15ng/ul | 18.11ng/ul |
| A260/A280 | 1.832 | 1.826 | 1.901 | 1.878 | 1.824 | 1.813 |
Example 5 Intelligent sample grinder extraction of DNA from 7 plant tissues
The intelligent sample grinder, the composition of the reagent and the extraction process are the same as those of the intelligent sample grinder in example 4 except for the grinding mode. The results are shown in Table 9 and FIG. 6. Therefore, the intelligent sample grinder can also achieve the purpose of extracting the DNA of the plant tissue by using the method.
Table 9: extraction of DNA quality of 7 plant tissues by intelligent sample grinder
Example 6 extraction of DNA from plant tissues with NaAc buffer at pH4.0 instead of Tris-HCl at pH8.0
The DNA of the scindapsus aureus leaf was extracted with NaAc at pH4.0 instead of Tris-HCl at pH8.0 and ground for 5 minutes. Wherein the first solution contains CTAB 3%, EDTA 20mM, NaCl 2.4M, glycerol 3%, NaAc 100mM, RNase 8ul 10mg/ml (added during DNA extraction). A second solution: 5M NaCl. Weighing scindapsus aureus leaf about 100 mg. The results were determined on a biaosharp (table 10) and fig. 7. It can be seen that Tris-HCl can be replaced by NaAc buffer.
Table 10: extraction of plant tissue DNA concentration with NaAc instead of Tris-HCl (100ul elution)
| Plant tissue name | Scindapsus aureus leaf |
| Quality of | 99.4mg |
| DNA concentration | 21.13ng/ul |
| A260/A280 | 1.817 |
Example 7 extraction of DNA from plant tissues with NaAc at pH7.0 or KAc buffer at pH7.0 instead of NaCl and KAc at pH7.2
The DNA of the leaf of Gloiopeltis aureus was extracted with NaAc at 5M pH7.0 or KAc at 5M pH7.0 instead of 5M NaCl and ground for 5 minutes. Wherein the first solution comprises CTAB 3%, EDTA 20mM, NaAc or KAc 2.4M, glycerol 3%, Tris-HCl 100mM, 8ul 10mg/ml RNase (added during DNA extraction). A second solution: NaAc at 5M pH7.0 or KAc at 5M pH 7.0. Weighing scindapsus aureus leaf about 200 mg. The results were determined on a biaosharp (table 11) and fig. 8. Therefore, NaAc or KAc can be used instead of NaCl to extract DNA from plant tissue.
Table 11: DNA concentration of plant tissue was extracted by using NaAc or KAc instead of NaCl and KAc at pH7.2 (100ul elution)
The result shows that the extraction method provided by the invention has better universality in the aspect of plant tissue DNA extraction, adopts common reagents/instruments in the field, is suitable for DNA extraction of various plant tissues, has higher DNA concentration and purity, and can be used for subsequent operations such as molecular cloning, sequencing and the like. More remarkably, the method has very short operation time, and provides more possibilities for shortening the whole experiment time and improving the experiment efficiency.
Sources of reagents used in the present invention:
EDTA national reagent CAS number: 6381-92-6
Tris-HCl national reagent CAS number: 77-86-1
NaCl national reagent CAS No.: 7647-14-5
The CAS number of the NaAc national reagent: 127-09-3
CAS number of KAc national medicine reagent: 127-08-2
CTAB national reagent CAS number: 57-09-0
Isopropyl alcohol national reagent CAS number: 67-63-0
Anhydrous ethanol national reagent CAS number: 64-17-5
RNase A Han remote International medicine science and technology (Beijing) Co., Ltd
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, combinations, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A method for extracting plant tissue DNA is characterized by comprising the following steps:
(a) adding plant tissue to a grinding device or grinder;
(b) pre-cooling the plant tissue and a grinding device, wherein the moisture of the plant tissue is condensed after the pre-cooling, the grinding device reaches a temperature after the pre-cooling, which enables the moisture to keep a condensed state when the plant tissue is ground therein, and the plant tissue is ground in a low-temperature drying state;
(c) grinding the plant tissue by using a pre-cooled grinding device under the condition of no protection of a liquid refrigerant to obtain plant tissue powder;
(d) and (3) carrying out DNA extraction on the plant tissue powder by using a CTAB cracking method.
2. The method for extracting plant tissue DNA according to claim 1, wherein: the pre-cooling in the step (b) is to add a liquid refrigerant to the grinding device carrying the plant tissues; the addition amount of the liquid refrigerant can enable the moisture of the plant tissue to be condensed after precooling, the grinding device to reach the temperature capable of keeping the moisture in a condensed state when the plant tissue is ground in the grinding device after precooling, and the liquid refrigerant volatilizes into a gaseous state to be dissipated after precooling the plant tissue and the grinding device.
3. The method for extracting plant tissue DNA according to claim 1, wherein: the grinding of step (c) is completed within 10 minutes, preferably within 5 minutes.
4. The method for extracting plant tissue DNA according to claim 1, wherein: step (d) further comprises:
i. transferring the plant tissue powder into a container containing the first solution and the RNase and mixing uniformly;
adding the second solution, uniformly mixing, centrifuging and retaining the supernatant;
adding the third solution, uniformly mixing, transferring into an adsorption column, centrifuging and removing the filtrate;
adding the fourth solution to the adsorption column, centrifuging and discarding the filtrate;
v. fully drying the adsorption column and transferring to a new container;
and vi, dropwise adding the fifth solution to the center of the adsorption column, standing at room temperature, centrifuging, and collecting filtrate.
5. The method for extracting plant tissue DNA according to claim 4, wherein: and steps i to vi are all completed in a room temperature environment.
6. The method for extracting plant tissue DNA according to claim 4, wherein: the first solution is a lysate comprising CTAB, Tris-HCl (or other buffers), EDTA, glycerol and NaCl (or other salts), 8ul of 10mg/ml RNase (added when extracting DNA); the concentration of CTAB is 2-5%, the concentration of Tris-HCl (or other buffer solution) is 50-100 mM, the concentration of EDTA is 10-20 mM, the concentration of glycerol is 1-4%, and the concentration of NaCl (or other salt) is 2.0-2.4M.
7. The method for extracting plant tissue DNA according to claim 4, wherein: the second solution is a NaCl (or other salt) solution with the concentration of 4-5M; the third solution is isopropanol, and the ratio of the addition amount of the isopropanol to the volume of the supernatant obtained in the step ii is 0.4-0.8: 1; the fourth solution is rinsing liquid containing 65-80% of ethanol; the fifth solution is TE eluent or pure water.
8. A kit for carrying out the method of any one of claims 1 to 7, the kit comprising: a first buffer solution containing 2-5% CTAB, 50-100 mM Tris-HCl (or other buffer solution), 10-20 mM EDTA, 1-4% glycerol, 2.0-2.4M NaCl (or other salt), and 8ul 10mg/ml RNase (added when extracting DNA); and a vector for describing the method according to any one of claims 1 to 7.
9. A computer readable carrier carrying a computer program comprising instructions for carrying out the method according to any one of claims 1 to 7.
10. An automated nucleic acid extraction device, the device comprising: a liquid filling/pipetting assembly, a centrifugation assembly, a grinding assembly and a controller electrically connected with the assemblies; the controller comprises the computer readable carrier of claim 9.
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