CN110951724A - Method for rapidly extracting nucleic acid DNA and/or RNA by Trizol - Google Patents
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Abstract
本发明公开了一种使用Trizol试剂快速提取核酸DNA和/或RNA的方法，可采用Trizol试剂和收集柱对样品进行DNA和RNA的分别提取或同时提取，相比于使用Trizol提取RNA和DNA的经典方法，提取时间短，稳定性好，可以满足不同试验对DNA和RNA的不同要求，非常有利于对材料中的DNA和RNA病原进行检测，同时也可以减少材料使用，对难得材料尤其重要。本发明提供的Trizol快速提取核酸DNA和/或RNA的方法，对于分子生物学试验具有重要的实用性。The invention discloses a method for rapidly extracting nucleic acid DNA and/or RNA by using Trizol reagent. Trizol reagent and collection column can be used to extract DNA and RNA from samples separately or simultaneously. Compared with the method of using Trizol to extract RNA and DNA The classic method, with short extraction time and good stability, can meet the different requirements of DNA and RNA in different experiments, which is very beneficial to the detection of DNA and RNA pathogens in materials, and can also reduce the use of materials, which is especially important for rare materials. The method for rapidly extracting nucleic acid DNA and/or RNA provided by Trizol in the present invention has important practicability for molecular biology experiments.
Description
Technical Field
The invention relates to the technical field of nucleic acid extraction, in particular to a method for rapidly extracting nucleic acid DNA and/or RNA by Trizol.
Background
Trizol reagent can rapidly extract total RNA of different tissues of human, animal, plant and bacteria, and the method is used for small amount of tissues (50-100mg) and cells (5X 10)6) And large amounts of tissue (. gtoreq.1 g) and cells (. gtoreq.1 g)>107) All have better separation effect. The simplicity of the Trizol reagent in operation allows multiple samples to be processed simultaneously. All operations can be completed in one hour. The total RNA extracted by the Trizol reagent can avoid the pollution of DNA and protein. Thus enabling northern blot analysis, dot blot hybridization, poly (A) + selection, in vitro translation, RNase protection analysis and molecular cloning. And the solubility of DNA, RNA and protein in different solutions can be utilizedThe efficiency is excellent when RNA (upper layer), DNA (middle layer) and protein (lower layer) in different layers are separated and purified by layering.
However, in the process of extracting RNA by Trizol reagent, it takes a long time for centrifugation to precipitate nucleic acid, and the precipitate is easily discarded when the supernatant is removed, resulting in poor stability of the extraction effect. In the process of extracting DNA, in addition to the above problems, repeated washing with an ethanol solution of sodium citrate is required, each washing time is long (30min), and time and labor are wasted.
Disclosure of Invention
The invention aims to provide a method for simply and rapidly extracting DNA and RNA from a sample respectively or simultaneously by using a Trizol reagent and a collecting column.
The invention is realized by the following technical scheme:
a method for Trizol to rapidly extract nucleic acid DNA and/or RNA is characterized in that,
1) the method for simultaneously extracting the DNA and the RNA comprises the following operation steps:
(1) putting fresh materials of which the amount is less than 50mg into a grinder, adding 0.5mL of Trizol reagent, quickly grinding and crushing, transferring into a centrifuge tube with 1.5mL of RNA enzyme removed, continuously reversing and uniformly mixing until the solution is clear, wherein the fresh materials comprise any one of tissues, cells, blood, plasma, secretion and culture solution;
(2) adding 600 μ L of separation liquid, wherein the separation liquid comprises 100-;
(3) transferring the solution after the oscillation and the uniform mixing into a collection column which is placed into a collection tube for 1min at 12000rpm for two times, and pouring out the liquid in the collection tube after each centrifugation;
(4) adding 600 mu L of the purified solution 1 into a collection column, wherein the purified solution 1 comprises 0.05-0.3M of sodium citrate and 5% -30% of DEPC water in ethanol, centrifuging at 12000rpm for 1min, pouring off the liquid in the collection column, then adding 600 mu L of the purified solution 1 into the collection column, centrifuging at 12000rpm for 1min, and pouring off the liquid in the collection column;
(5) adding 500 μ L of purified solution 2 into the collection column, wherein the 2-position of the purified solution contains DEPC water containing 70% -80% ethanol, and centrifuging at 12000rpm for 2 min;
(6) carefully taking out the collecting tube, transferring the collecting column to a new centrifuge tube, adding 100 mu L DEPC water to dissolve for 10min, and centrifuging at 12000rpm for 2min to obtain a nucleic acid solution containing DNA and RNA;
2) the RNA extraction method comprises the following operation steps:
(1) putting fresh materials of which the amount is less than 50mg into a grinder, adding 0.5mL of Trizol reagent, quickly grinding and crushing, transferring into a centrifuge tube with 1.5mL of RNA enzyme removed, continuously reversing and uniformly mixing until the solution is clear, wherein the fresh materials comprise any one of tissues, cells, blood, plasma, secretion and culture solution;
(2) adding 100 μ L chloroform, shaking, standing at room temperature for 5min, and centrifuging at 12000rpm for 5 min;
(3) transferring the upper water phase into a collecting column placed in a collecting pipe, centrifuging at 12000rpm for 1min, and pouring out liquid in the collecting pipe;
(4) adding 600 mu L of purified liquid 1 into a collection column, centrifuging at 12000rpm for 1min, pouring out the liquid in the collection tube, then adding 600 mu L of purified liquid 1 into the collection column, centrifuging at 12000rpm for 1min, and pouring out the liquid in the collection tube;
(5) adding 500 μ L of purified solution 2 into the collection column, and centrifuging at 12000rpm for 2 min;
(6) carefully taking out the collecting tube, transferring the collecting column to a new centrifuge tube, adding 100 mu L DEPC water to dissolve for 10min, and centrifuging at 12000rpm for 2min to obtain a nucleic acid solution containing RNA;
3) the method for extracting the DNA comprises the following operation steps:
(1) placing fresh materials less than 50mg in a grinder, adding 0.5mL Trizol reagent, quickly grinding and crushing, transferring to a 1.5mL centrifuge tube without RNase, and continuously reversing and uniformly mixing until the materials are clear; adding 100 μ L chloroform, shaking, standing at room temperature for 5min, and centrifuging at 12000rpm for 5min, wherein the fresh material comprises any one of tissue, cell, blood plasma, secretion, and culture solution;
(2) removing upper water phase, reversing, mixing lower solution, adding 300 μ L of separation solution composed of 50-100 μ L chloroform, 50-150 μ L ethanol, and 50-100 μ L isopropanol, shaking, and mixing;
(3) transferring the solution into a collecting column placed in a collecting pipe, centrifuging at 12000rpm for 1min, and then pouring out the liquid in the collecting pipe.
(4) Adding 600 mu L of purified liquid 1 into a collection column, centrifuging at 12000rpm for 1min, pouring out the liquid in the collection tube, then adding 600 mu L of purified liquid 1 into the collection column, centrifuging at 12000rpm for 1min, and pouring out the liquid in the collection tube;
(5) adding 500 μ L of purified solution 2 into the collection column, and centrifuging at 12000rpm for 2 min;
(6) the collection tube was carefully removed, the collection column was transferred to a new centrifuge tube, and 100. mu.L ddH was added2O or TE buffer solution, after 10min of dissolution, 12000rpm centrifugation for 2min, containing DNA nucleic acid solution.
According to the technical scheme, the beneficial effects of the invention are as follows:
1. the Trizol and the collecting column can be used for simultaneously extracting or respectively extracting DNA and RNA, and the requirements of various molecular biological detections such as various amplifications, hybridization and the like can be met.
2. The extraction time is short, the time for extracting nucleic acid by using Trizol can be reduced by half or even two thirds, and the extraction can be completed within 30 min.
3. Good stability, difficult loss of nucleic acid in the extraction process, very stable extraction effect and good uniformity.
4. The sample requirement is low, DNA and RNA can be extracted simultaneously by using one sample material, the material usage amount is reduced, and the method is particularly important for difficult materials.
5. Does not need a water bath and a refrigerated centrifuge, has wider applicability and is convenient to popularize in the basic level.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
Extraction of attenuated vaccine nucleic acid for porcine respiratory and reproductive syndrome
1. Sucking 50 mu L of diluted vaccine solution, putting the diluted vaccine solution into a 1.5mL centrifuge tube without RNA enzyme, adding 0.5mL of TRIZOL, and continuously reversing and uniformly mixing until the diluted vaccine solution is clear;
2. adding 100 μ L chloroform, shaking, standing at room temperature for 3min, and centrifuging at 12000g for 5 min;
3. transferring the upper water phase into a collecting column placed in a collecting pipe, centrifuging at 12000rpm for 1min, and pouring out liquid in the collecting pipe;
4. adding 600 mu L of purified liquid 1 into a collection column, wherein the purified liquid 1 comprises 0.05M of sodium citrate and 5% of ethanol DEPC water, centrifuging at 12000rpm for 1min, pouring out the liquid in the collection column, adding 600 mu L of purified liquid 1 into the collection column, wherein the purified liquid 1 contains 0.05M of sodium citrate and 5% of ethanol DEPC water, centrifuging at 12000rpm for 1min, and pouring out the liquid in the collection column;
5. adding 500 μ L of purified solution 2 into the collection column, wherein the purified solution 2 is DEPC water containing 60% ethanol, and centrifuging at 12000rpm for 2 min;
6. carefully taking out the collecting tube, transferring the collecting column to a new centrifuge tube, adding 100 mu L DEPC water to dissolve for 10min, and then centrifuging at 12000rpm for 2min to obtain the nucleic acid solution containing the porcine respiratory and reproductive syndrome virus RNA.
Example 2
Extraction of pseudorabies virus attenuated vaccine nucleic acid
1. Sucking 50 mu L of diluted pseudorabies attenuated vaccine solution, putting the pseudorabies attenuated vaccine solution into a 1.5mL centrifuge tube, adding 0.5mL Trizol, continuously reversing and uniformly mixing until the pseudorabies attenuated vaccine solution is clear; adding 100 μ L chloroform, shaking, standing at room temperature for 5min, and centrifuging at 12000rpm for 5 min;
2. removing upper water phase, reversing, mixing lower solution, adding 300 μ L of separation solution composed of 100 μ L chloroform, 100 μ L ethanol, and 100 μ L isopropanol, and shaking;
3. transferring the solution into a collecting column placed in a collecting pipe, centrifuging at 12000rpm for 1min, and then pouring out the liquid in the collecting pipe.
4. Adding 600 mu L of purified liquid 1 into a collection column, wherein the purified liquid 1 comprises 0.2M sodium citrate and 25% ethanol DEPC water, centrifuging at 12000rpm for 1min, pouring out the liquid in the collection column, adding 600 mu L of purified liquid 1 into the collection column, wherein the purified liquid 1 comprises 0.2M sodium citrate and 25% ethanol DEPC water, centrifuging at 12000rpm for 1min, and pouring out the liquid in the collection column;
5. adding 500 μ L of purified solution 2 into the collection column, wherein the purified solution 2 contains 70% ethanol DEPC water, centrifuging at 12000rpm for 2 min;
6. the collection tube was carefully removed, the collection column was transferred to a new centrifuge tube, and 100. mu.L ddH was added2Dissolving in O or TE buffer solution for 10min, and centrifuging at 12000rpm for 2min to obtain nucleic acid solution containing pseudorabies virus DNA.
Example 3
Simultaneous extraction of DNA and RNA from porcine tissue
1. Taking 50mg of fresh tissue of the pig, grinding the tissue by using liquid nitrogen, transferring the ground tissue to a 1.5mL centrifuge tube without RNase, adding 0.5mL of TRIZOL, continuously reversing and uniformly mixing the tissue until the tissue is clear;
(2) adding 600 μ L of separation solution composed of 200 μ L chloroform, 300 μ L ethanol, and 100 μ L isopropanol, and shaking;
(3) transferring the solution after the oscillation and the uniform mixing into a collecting column which is placed into a collecting pipe for two times, centrifuging at 12000rpm for 1min, and pouring out liquid in the collecting pipe;
(4) adding 600 mu L of purified liquid 1 into a collection column, wherein the purified liquid 1 comprises 0.3M sodium citrate and 30% ethanol DEPC water, centrifuging at 12000rpm for 1min, pouring off the liquid in the collection column, adding 600 mu L of purified liquid 1 into the collection column, wherein the purified liquid 1 comprises 0.3M sodium citrate and 30% ethanol DEPC water, centrifuging at 12000rpm for 1min, and pouring off the liquid in the collection column;
(5) adding 500 μ L of purified solution 2 into the collection column, wherein the purified solution 2 is DEPC water containing 80% ethanol, and centrifuging at 12000rpm for 2 min;
(6) the collection tube was carefully removed, the column was transferred to a new rnase-free centrifuge tube, dissolved in 100. mu.L EPC water for 10min and centrifuged at 12000rpm for 2min to obtain a nucleic acid solution containing porcine DNA and RNA (including DNA and RNA infected with pathogens in porcine tissue).
The embodiment in the application improves the method for extracting nucleic acid by Trizol, has short extraction time and good stability, can simultaneously or respectively extract DNA and RNA, meets different requirements of different tests on the DNA and the RNA, is very favorable for detecting the DNA and the RNA pathogens in the pathological materials, is also favorable for reducing the use of the pathological materials, and is particularly important for rare materials.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (2)
1. A method for Trizol to rapidly extract nucleic acid DNA and/or RNA is characterized in that,
1) the method for simultaneously extracting the DNA and the RNA comprises the following operation steps:
(1) placing less than 50mg of fresh materials into a grinder, adding 0.5mL of Trizol reagent, quickly grinding and crushing, transferring into a 1.5mL centrifuge tube without RNase, and continuously reversing and uniformly mixing until the solution is clear;
(2) adding 600 μ L of separation liquid, wherein the separation liquid comprises 100-;
(3) transferring the solution after the oscillation and the uniform mixing into a collection column which is placed into a collection tube for 1min at 12000rpm for two times, and pouring out the liquid in the collection tube after each centrifugation;
(4) adding 600 mu L of the purified solution 1 into a collection column, wherein the purified solution 1 comprises 0.05-0.3M of sodium citrate and 5% -30% of DEPC water in ethanol, centrifuging at 12000rpm for 1min, pouring off the liquid in the collection column, then adding 600 mu L of the purified solution 1 into the collection column, centrifuging at 12000rpm for 1min, and pouring off the liquid in the collection column;
(5) adding 500 μ L of purified solution 2 into the collection column, wherein the purified solution 2 is DEPC water containing 70% -80% ethanol, and centrifuging at 12000rpm for 2 min;
(6) carefully taking out the collecting tube, transferring the collecting column to a new centrifuge tube, adding 100 mu L DEPC water to dissolve for 10min, and centrifuging at 12000rpm for 2min to obtain a nucleic acid solution containing DNA and RNA;
2) the RNA extraction method comprises the following operation steps:
(1) placing less than 50mg of fresh materials into a grinder, adding 0.5mL of Trizol reagent, quickly grinding and crushing, transferring into a 1.5mL centrifuge tube without RNase, and continuously reversing and uniformly mixing until the solution is clear;
(2) adding 100 μ L chloroform, shaking, standing at room temperature for 5min, and centrifuging at 12000rpm for 5 min;
(3) transferring the upper water phase into a collecting column placed in a collecting pipe, centrifuging at 12000rpm for 1min, and pouring out liquid in the collecting pipe;
(4) adding 600 mu L of purified liquid 1 into a collection column, centrifuging at 12000rpm for 1min, pouring out the liquid in the collection tube, then adding 600 mu L of purified liquid 1 into the collection column, centrifuging at 12000rpm for 1min, and pouring out the liquid in the collection tube;
(5) adding 500 μ L of purified solution 2 into the collection column, and centrifuging at 12000rpm for 2 min;
(6) carefully taking out the collecting tube, transferring the collecting column to a new centrifuge tube, adding 100 mu L DEPC water to dissolve for 10min, and centrifuging at 12000rpm for 2min to obtain a nucleic acid solution containing RNA;
3) the method for extracting the DNA comprises the following operation steps:
(1) placing fresh materials less than 50mg in a grinder, adding 0.5mL Trizol reagent, quickly grinding and crushing, transferring to a 1.5mL centrifuge tube without RNase, and continuously reversing and uniformly mixing until the materials are clear; adding 100 μ L chloroform, shaking, standing at room temperature for 5min, and centrifuging at 12000rpm for 5 min;
(2) removing upper water phase, reversing, mixing lower solution, adding 300 μ L of separation solution composed of 50-100 μ L chloroform, 50-150 μ L ethanol, and 50-100 μ L isopropanol, shaking, and mixing;
(3) transferring the solution into a collecting column placed in a collecting pipe, centrifuging at 12000rpm for 1min, and then pouring out the liquid in the collecting pipe.
(4) Adding 600 mu L of purified liquid 1 into a collection column, centrifuging at 12000rpm for 1min, pouring out the liquid in the collection tube, then adding 600 mu L of purified liquid 1 into the collection column, centrifuging at 12000rpm for 1min, and pouring out the liquid in the collection tube;
(5) adding 500 μ L of purified solution 2 into the collection column, and centrifuging at 12000rpm for 2 min;
(6) the collection tube was carefully removed, the collection column was transferred to a new centrifuge tube, and 100. mu.L ddH was added2O or TE buffer solution, after 10min of dissolution, 12000rpm centrifugation for 2min, containing DNA nucleic acid solution.
2. The method for Trizol rapid extraction of nucleic acid DNA and/or RNA as claimed in claim 1, wherein the fresh material comprises any one of tissue, cell, blood, plasma, secretion and culture solution.
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Cited By (2)
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| CN115287281A (en) * | 2022-05-30 | 2022-11-04 | 华南农业大学 | A kind of total DNA extraction method of sea cucumber |
| CN117625740A (en) * | 2023-11-01 | 2024-03-01 | 嘉应学院 | A method to rapidly extract DNA from Trizol samples suitable for high-throughput sequencing |
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