Rat tail lysate and rat tail rapid PCR amplification sequencing detection method

Abstract

The invention discloses a rat tail lysate and a rat tail rapid PCR amplification sequencing detection method, the rat tail lysate comprises the following components: 20-200 mmol / L of sodium hydroxide, 2-5 mol / L of isohydrosulfate, 1.5-3 mol / L of guanidine hydrochloride, 100-500 mmol / L of sodium chloride and 1-3 mol / L. In addition, the invention also provides the rat tail rapid PCR amplification sequencing detection method, and the rat tail lysate comprises the following components: 20-200 mmol / L of sodium hydroxide, 2-5 mol / L of isohydrosulfate, 1.5-3 mol / L of guanidine hydrochloride, 100-500 mmol / L of sodium chloride and 1-3 mol / L of ethylenediamine tetraacetic acid. When the rat tail lysate is used for lysing, the obtained supernate is directly subjected to PCR amplification without centrifugation, so that the problems that the required nucleic acid product cannot be quickly obtained from the current rat tail lysate and is directly used for subsequent PCR amplification, and the high-throughput operation is limited are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA extraction, and particularly to a mouse tail lysis solution and a rapid PCR amplification and sequencing detection method for mouse tails. Background Art

[0002] Mice are highly similar to humans in terms of genetics, physiology, and development, and are convenient for laboratory breeding and management, thus having extensive applications in scientific research.

[0003] In recent years, with the rapid development of the CRISPR / Cas9 gene editing technology, the CRISPR / Cas9 technology has been increasingly used to perform gene editing on mice, followed by screening and identifying gene editing mutants for corresponding research. Among them, nucleic acid extraction, as a key step in the screening process, has an impact on the rapid identification of gene-edited mice and the progress of research to a certain extent. Specifically, the tail of the corresponding mouse is lysed to obtain nucleic acid products, and then the obtained nucleic acid products are subjected to PCR (Polymerase Chain Reaction) amplification, that is, polymerase chain reaction amplification, and then compared and identified.

[0004] Most of the existing methods for mouse tail lysis are grinding, using mouse tail lysis solution or enzyme treatment. However, the existing mouse tail lysis solutions have the problems of long time consumption and cumbersome operation, and need to be further purified before being used for PCR amplification. They cannot quickly obtain the required nucleic acid products and directly use them for PCR amplification. In addition, the existing mouse tail lysis solutions need to be combined with multiple centrifugations, and the throughput is limited by the centrifuge, resulting in limited high-throughput operation. Summary of the Invention

[0005] Aiming at the above defects, the purpose of the present invention is to provide a mouse tail lysis solution and a rapid PCR amplification and sequencing detection method for mouse tails, which are applicable to experimental mice and do not require additional purification steps, solving the problems that the existing mouse tail lysis solutions cannot quickly obtain the required nucleic acid products and directly use them for subsequent PCR amplification, and the high-throughput operation is limited.

[0006] To achieve this purpose, the present invention adopts the following technical solutions:

[0007] A mouse tail lysis solution, comprising the following components: sodium hydroxide at 20 - 200 mmol / L, isothiocyanate at 2 - 5 mol / L, guanidine hydrochloride at 1.5 - 3 mol / L, sodium chloride at 100 - 500 mmol / L, and ethylenediaminetetraacetic acid at 1 - 3 mol / L.

[0008] Preferably, the mouse tail lysis solution comprises the following components: sodium hydroxide at 150 mmol / L, isothiocyanate at 4 mol / L, guanidine hydrochloride at 2 mol / L, sodium chloride at 300 mmol / L, and ethylenediaminetetraacetic acid at 2 mol / L.

[0009] A rapid PCR amplification and sequencing detection method for mouse tails, comprising the following steps:

[0010] S1. Take mouse tail samples and place them in different wells of a PCR plate. Add the mouse tail lysate described in any one of claims 1-2 to soak the mouse tail samples in each well;

[0011] S2. Place the PCR plate containing the mouse tail lysate and mouse tail samples in a PCR instrument, and heat and incubate until complete lysis;

[0012] S3. Use a multi-channel pipette to transfer the supernatant to a new PCR plate for storage;

[0013] S4. Take the supernatant for PCR amplification.

[0014] Further, in step S1, the addition amount of the mouse tail lysate is 50-150 μl.

[0015] Preferably, in step S1, the length of the mouse tail sample is 1-2 mm.

[0016] Preferably, in step S2, incubate at 95-100 °C for 15 min-30 min.

[0017] Preferably, in step S4, take 2 μl of the supernatant and place it in a new PCR plate for PCR amplification.

[0018] Preferably, in step S4, the PCR amplification system is 2 μl of the supernatant, 25 μl of 2x Taq Mix, and a primer set of 20 μmol / L. Add sterile water to make the total volume of the reaction system 50 μl;

[0019] The reaction program for PCR amplification is: 95 °C for 3 minutes; 95 °C for 15 seconds, 60 °C for 15 seconds, 72 °C for 30 seconds, 33 cycles; 72 °C for 5 minutes.

[0020] The technical solution provided by the present invention may include the following beneficial effects:

[0021] Isothiocyanate and guanidine hydrochloride are added to the mouse tail lysate, which cooperate with the added sodium hydroxide and ethylenediaminetetraacetic acid to ensure the stability and efficiency of the overall lysis. At the same time, it prevents nucleic acids from being degraded during lysis, and quickly obtains the required nucleic acid products. In addition, isothiocyanate can bind to impurities such as proteins, reducing the centrifugation step. The obtained supernatant can be directly used for subsequent PCR amplification, enabling high-throughput experiments. Description of the Drawings

[0022] Figure 1 It is the electrophoresis result diagram of Example Groups 1-4 of the present invention.

[0023] Figure 2 It is the electrophoresis result diagram of Example Group 5 of the present invention. Detailed implementation manners

[0024] The technical solution of the present invention will be further described below through specific implementation manners.

[0025] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0026] For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments not specified in terms of the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0027] A mouse tail lysate, comprising the following components: sodium hydroxide at 20 - 200 mmol / L, isothiocyanate at 2 - 5 mol / L, guanidine hydrochloride at 1.5 - 3 mol / L, sodium chloride at 100 - 500 mmol / L, and ethylenediaminetetraacetic acid at 1 - 3 mol / L.

[0028] With the rapid development of the CRISPR / Cas9 gene editing technology, more and more CRISPR / Cas9 technology is used to perform gene editing on mice. To screen out the required mutants from multiple gene - edited mutant mice, it is necessary to cut the mouse tail for lysis to obtain nucleic acid products for subsequent identification or sequencing work.

[0029] The lysis methods of mouse tail tissue are mainly divided into physical method, chemical method, and enzymatic treatment method. The physical method usually uses mechanical means such as grinding to break the tissue cells by external force to release the components inside the cells; the enzymatic treatment method mainly uses the specific action of enzymes such as proteinase K to decompose the protein components in the cell structure and gently achieve cell lysis, while the chemical method uses a lysate, relying on the penetration and dissolution of its chemical components, to rupture the cell membrane and then release the cell contents. Compared with the physical method and the enzymatic treatment method, using the lysate is more simple in operation and causes less damage to cells.

[0030] However, in the prior art, the lysis solution prepared by the chemical method usually requires the use of a centrifuge because the existing chemical lysis solution has limited lysis effect on mouse tail tissue blocks. A centrifuge is needed for solid-liquid separation to increase the concentration of the nucleic acid product obtained by lysis and reduce the influence of other impurities on subsequent PCR amplification. It is impossible to quickly obtain the required nucleic acid product and directly use it for subsequent PCR amplification. Therefore, the present invention provides a mouse tail lysis solution applicable to experimental mice, which comprises five components. The three main components are sodium hydroxide at a concentration of 20-200 mmol / L, isothiocyanate at a concentration of 2-5 mol / L, and guanidine hydrochloride at a concentration of 1.5-3 mol / L. The combined action of the above three components is to dissolve the cell membrane and nuclear membrane, denature proteins, and release nucleic acids. Among them, sodium hydroxide can efficiently and fully lyse mouse tail tissue blocks at high temperature. Compared with the mouse tail lysis solution on the market that requires dilute hydrochloric acid of a standard concentration, sodium hydroxide is easier to prepare; both isothiocyanate and guanidine hydrochloride are strong protein denaturants, which can quickly dissolve the proteins inside the cells, destroy the cell structure, and separate nucleic acids from proteins. Isothiocyanate has the strongest denaturing ability among common protein denaturants, while guanidine hydrochloride is an efficient nuclease inhibitor, which can protect nucleic acid products from degradation. While heating and lysing mouse tail tissue, the three can dissolve nuclear proteins to separate them from nucleic acid DNA molecules, ensuring the integrity of the primary structure of nucleic acids, and thus ensuring the integrity of DNA templates in subsequent PCR amplification. The existing mouse tail lysis solution generally directly uses guanidine isothiocyanate, but it is unstable in solution, needs to be protected from light, and is prone to precipitation, resulting in reduced effects. The other two components are sodium chloride at a concentration of 100-500 mmol / L and ethylenediaminetetraacetic acid (EDTA) at a concentration of 1-3 mol / L. EDTA is a metal chelating agent that can chelate metal cations and inhibit nuclease activity, thereby inhibiting the degradation of genomic DNA by intracellular DNA enzymes. At the same time, sodium chloride provides positively charged sodium ions, and the sodium ions will combine with the released negatively charged DNA, thereby increasing the stability of DNA. Compared with directly using EDTA and sodium hydroxide, the present invention adds isothiocyanate and guanidine hydrochloride, both of which have strong penetration ability and protein denaturing effects, can quickly destroy the cell membrane and cell wall structures, accelerate the lysis process, and inhibit the activity of DNA enzymes to make up for the deficiency of the low lysis efficiency of sodium hydroxide, ensuring the stability and efficiency of overall lysis. At the same time, it jointly inhibits nuclease activity with EDTA to prevent nucleic acids from being degraded during the lysis process.

[0031] In addition, sodium hydroxide can rapidly lyse cells, but its strong alkalinity may cause nucleic acid degradation. Therefore, the concentration of sodium hydroxide is limited to 20-200 mmol / L, and 2-5 mol / L of isothiocyanate and 1.5-3 mol / L of guanidine hydrochloride are added. The addition of isothiocyanate and guanidine hydrochloride at corresponding concentrations can buffer this strong alkaline environment and reduce the damage to nucleic acids. If it is less than this range, the concentration is too low and the lysis effect is poor; if it is greater than this range, it will affect the integrity of DNA. It should be noted that isothiocyanate can bind to impurities such as proteins and precipitate them, thereby improving the purity of nucleic acid extraction, protecting the integrity of nucleic acids and reducing impurity interference. Without additional purification steps, it can directly perform subsequent PCR amplification, simplify the experimental process, and solve the problem that the existing mouse tail lysis solution cannot quickly obtain the required nucleic acid products and directly use them for subsequent PCR amplification.

[0032] Preferably, the mouse tail lysis solution comprises the following components: 150 mmol / L of sodium hydroxide, 4 mol / L of isothiocyanate, 2 mol / L of guanidine hydrochloride, 300 mmol / L of sodium chloride, and 2 mol / L of ethylenediaminetetraacetic acid.

[0033] Preferably, under the limitation of the concentrations of the above components, the obtained mouse tail lysis solution has the best lysis effect and can effectively bind impurities such as proteins, reducing the impact on subsequent PCR amplification. While having amplification bands, it has sufficient brightness, that is, after lysing mouse tail tissue with the mouse tail lysis solution at this concentration, the obtained DNA concentrations are relatively consistent and the purity is high. When performing subsequent PCR amplification, the brightness of the obtained electrophoresis results is uniform and obvious, and the effect is better.

[0034] A method for rapid PCR amplification and sequencing detection of mouse tails comprises the following steps:

[0035] S1. Take mouse tail samples and place them in different wells of a PCR plate respectively, and add the mouse tail lysis solution according to any one of claims 1-2 to soak the mouse tail samples in each well;

[0036] S2. Place the PCR plate containing the mouse tail lysis solution and the mouse tail samples in a PCR instrument, and heat and incubate until sufficient lysis;

[0037] S3. Use a multi-channel pipette to transfer the supernatant to a new PCR plate for storage;

[0038] S4. Take the supernatant for PCR amplification.

[0039] In the prior art, in the method of using mouse tail lysis solution for lysis and performing rapid PCR amplification and sequencing detection, an EP tube is generally used as the reaction container because centrifugation is required later. However, in the present invention, a PCR plate is directly used as the reaction container. Since the addition of isothiocyanate can bind to impurities such as proteins and cause them to precipitate, the purity of nucleic acid extraction can be improved, the integrity of nucleic acid can be protected, and interference from impurities can be reduced. No additional purification step is required, the centrifugation process can be omitted, and a PCR plate can be directly used as the reaction container. Subsequently, the liquid transfer steps can be reduced, and PCR identification can be carried out quickly.

[0040] In addition, when a multi-channel pipette is used in combination with a multi-well PCR plate, for example, in combination with a 96-well PCR plate, high-throughput operation can be effectively carried out without being restricted by a centrifuge. If restricted by a centrifuge, at most 24 samples can be processed at one time, and the high-throughput requirement cannot be achieved. The mouse tail lysis solution of the present invention has a relatively fast lysis speed and requires less lysis time, solving the problem that the existing mouse tail lysis solution is limited in high-throughput experiments.

[0041] Preferably, in step S1, the addition amount of the mouse tail lysis solution is 50 - 150 μl.

[0042] Specifically, in step S1, it is necessary to add the mouse tail lysis solution to cover the mouse tail sample, but the addition amount of the mouse tail lysis solution will affect the subsequent PCR amplification result. Within this defined range, it can ensure a more reasonable ratio between the mouse tail tissue and the mouse tail lysis solution, enable the mouse tail lysis solution to penetrate the tissue more efficiently, accelerate the cell lysis process, effectively digest the mouse tail, have an appropriate amount of lysed cells, and at the same time not dilute the amount of lysed cells, ensuring the DNA yield, helping to better protect the integrity of DNA, avoiding DNA degradation caused by excessive mouse tail lysis solution, thereby providing a high-quality template for the subsequent PCR reaction and ensuring the accuracy of the subsequent PCR experimental results.

[0043] In addition, the usage amount of reagents can be reduced, the experimental cost can be lowered, and at the same time, the generation of waste can be reduced.

[0044] Further, in step S1, the length of the mouse tail sample is 1 - 2 mm.

[0045] It should be noted that the sensitivity of the PCR reaction refers to the minimum number of target molecules that can be detected in each PCR reaction tube. In theory, a single copy of the target gene can be detected at the lowest in each PCR reaction tube. However, affected by factors such as the interfering substances in the PCR reaction of the extracted DNA template, the number of target molecules required to actually detect the amplification product is often relatively high. Since the volume of the template added to the PCR reaction is limited, the better the lysis degree of the mouse tail tissue block, the higher the content of nucleic acid DNA molecules in each microliter of the sample. And the present invention only needs to use a 1-2 mm mouse tail tissue, and the volume of the added mouse tail lysate is small, 50-150 μl. In this way, while obtaining a higher concentration of DNA template, the required lysis time will be less, the lysis efficiency is higher, without affecting the effect of subsequent PCR amplification, and at the same time reducing the harm to the mice.

[0046] In addition, the length of the mouse tail sample is 1-2 mm. The present invention does not require centrifugation. A smaller tissue sample volume can reduce the impurity content in the supernatant, thereby reducing the inhibitory effect on the subsequent PCR reaction, enabling the mouse tail lysate to effectively lyse the mouse tail sample and having a high DNA yield, ensuring the accuracy of the subsequent PCR experimental results.

[0047] Preferably, in step S2, incubate at 95-100 °C for 15 min-30 min.

[0048] Specifically, in order to make the cells lysed more fully, the sample is generally heated at a high temperature during the lysis process. The limited temperature is 95-100 °C, which can quickly destroy the cell membrane and cell wall, accelerate the dissolution of the tissue and the release of DNA, significantly shorten the lysis time, ensure sufficient reaction of the mouse tail tissue, and have a high DNA yield. At the same time, it does not affect the integrity of the genome. In addition, it can ensure the high efficiency of the operation, without the need for multiple time changes, reducing the operation time, that is, the heating and cooling time of the PCR instrument. And high-temperature incubation can precipitate the impurities in the mouse tail tissue, reduce the interference with the subsequent PCR reaction, and improve the specificity and success rate of PCR.

[0049] In addition, incubating at this temperature for 15 min-30 min ensures sufficient reaction of the mouse tail tissue, and at the same time reduces the damage to the integrity of genomic DNA caused by continuous high temperature.

[0050] Preferably, in step S4, take 2 μl of the supernatant into a new PCR plate for PCR amplification.

[0051] In a general PCR reaction system, the added amount of DNA template is 10-200 ng. Excessive DNA template will inhibit the progress of the polymerase chain reaction. After the mouse tail lysate of the present invention lyses the corresponding mouse tail sample, in a volume of 2 μl, the amount of DNA contained is more appropriate, ensuring the accuracy of the results obtained from the subsequent PCR reaction.

[0052] Preferably, in step S4, the PCR amplification system is 2 μl of supernatant, 25 μl of 2x Taq Mix, and a primer set of 20 μmol / L. Sterile water is added to make the total volume of the reaction system 50 μl.

[0053] The reaction program for PCR amplification is: 95°C for 3 minutes; 95°C for 15 seconds, 60°C for 15 seconds, 72°C for 30 seconds, for 33 cycles; 72°C for 5 minutes.

[0054] Specifically, the 2x Taq Mix contains Taq DNA polymerase, magnesium chloride, and dNTPs. The primer set is a forward primer of 10 μmol / L and a reverse primer of 10 μmol / L. The primer set does not specifically refer to primers of a certain sequence, but is a primer designed for the current mouse tail sample-related PCR amplification operation, and needs to be designed according to the position of the gene editing target site and the knockout size. The specific primer design method can refer to the book "Molecular Biology Experimental Techniques".

[0055] Under this system and reaction program, the specificity of the reaction can be significantly improved, non-specific amplification can be reduced, thereby improving the accuracy of detection. And defining the reaction system can ensure the consistency of each experimental condition, reduce experimental errors caused by reagent concentration differences or reaction condition fluctuations, which is suitable for the purpose of high-throughput experiments.

[0056] The technical solution of the present invention will be further described below through specific embodiments.

[0057] Example group

[0058] Example group 1

[0059] S1. Take 4 pieces of 2 mm mouse tail samples with forceps and place them into wells A1 - A4 of a 96-well PCR plate. Add 50 μl of mouse tail lysis solution to each of wells A1 - A4 to cover the mouse tail samples. The components of the mouse tail lysis solution are 100 mmol / L sodium hydroxide, 2 mol / L isothiocyanate, 1.5 mol / L guanidine hydrochloride, 200 mmol / L sodium chloride, and 1.5 mol / L ethylenediaminetetraacetic acid.

[0060] S2. Place the 96-well PCR plate containing the mouse tail lysis solution and mouse tail samples in a PCR instrument and incubate at 95°C for 30 min.

[0061] S3. Use a multi-channel pipette to transfer the supernatant to a new PCR plate for storage.

[0062] S4. Take the supernatant from the new PCR plate containing the supernatant as the PCR template, and perform PCR amplification according to the following system and program;

[0063] The PCR amplification system consisted of 2 μl of supernatant, 25 μl of 2x Taq Mix, and a primer set at 20 μmol / L. Sterile water was added to make the total volume of the reaction system 50 μl.

[0064] The PCR amplification reaction program was as follows: 95°C for 3 minutes; 95°C for 15 seconds, 60°C for 15 seconds, 72°C for 30 seconds, for 33 cycles; 72°C for 5 minutes.

[0065] S5. Take 10 μl of the obtained PCR product and run agarose gel electrophoresis.

[0066] Example Group 2

[0067] S1. Take 4 pieces of 2-mm mouse tail samples with forceps and place them into wells A5 - A8 of a 96-well PCR plate. Add 50 μl of mouse tail lysis solution to each of wells A5 - A8 to cover the mouse tail samples. The components of the mouse tail lysis solution were 100 mmol / L sodium hydroxide, 4 mol / L isothiocyanate, 2 mol / L guanidine hydrochloride, 300 mmol / L sodium chloride, and 2 mol / L ethylenediaminetetraacetic acid.

[0068] S2. Place the 96-well PCR plate containing the mouse tail lysis solution and mouse tail samples in a PCR instrument and incubate at 95°C for 30 min.

[0069] S3. Use a multi-channel pipette to transfer the supernatant to a new PCR plate for storage.

[0070] S4. Use a multi-channel pipette to take the supernatant from the new PCR plate containing the supernatant as the PCR template and perform PCR amplification according to the following system and program;

[0071] The PCR amplification system consisted of 2 μl of supernatant, 25 μl of 2x Taq Mix, and a primer set at 20 μmol / L. Sterile water was added to make the total volume of the reaction system 50 μl.

[0072] The PCR amplification reaction program was as follows: 95°C for 3 minutes; 95°C for 15 seconds, 60°C for 15 seconds, 72°C for 30 seconds, for 33 cycles; 72°C for 5 minutes.

[0073] S5. Take 10 μl of the obtained PCR product and run agarose gel electrophoresis.

[0074] Example Group 3

[0075] S1. Take four 2-mm mouse tail samples respectively with forceps and clamp them into wells B1 - B4 of a 96-well PCR plate. Add 50 μl of mouse tail lysis solution to each of wells B1 - B4 to cover the mouse tail samples. The components of the mouse tail lysis solution are 150 mmol / L sodium hydroxide, 2 mol / L isothiocyanate, 1.5 mol / L guanidine hydrochloride, 200 mmol / L sodium chloride, and 1.5 mol / L ethylenediaminetetraacetic acid;

[0076] S2. Place the 96-well PCR plate containing the mouse tail lysis solution and mouse tail samples in a PCR instrument and incubate at 95 °C for 30 min;

[0077] S3. Use a multi-channel pipette to transfer the supernatant to a new PCR plate for storage;

[0078] S4. Use a multi-channel pipette to take the supernatant from the new PCR plate containing the supernatant as the PCR template and perform PCR amplification according to the following system and procedure;

[0079] The system for PCR amplification is 2 μl of supernatant, 25 μl of 2x Taq Mix, and a primer set of 20 μmol / L. Add sterile water to make the total volume of the reaction system 50 μl;

[0080] The reaction procedure for PCR amplification is: 95 °C for 3 minutes; 95 °C for 15 seconds, 60 °C for 15 seconds, 72 °C for 30 seconds, for 33 cycles; 72 °C for 5 minutes.

[0081] S5. Take 10 μl of the obtained PCR product and run agarose gel electrophoresis.

[0082] Example Group 4

[0083] S1. Take four 2-mm mouse tail samples respectively with forceps and clamp them into wells B5 - B8 of a 96-well PCR plate. Add 50 μl of mouse tail lysis solution to each of wells B5 - B8 to cover the mouse tail samples. The components of the mouse tail lysis solution are 150 mmol / L sodium hydroxide, 4 mol / L isothiocyanate, 2 mol / L guanidine hydrochloride, 300 mmol / L sodium chloride, and 2 mol / L ethylenediaminetetraacetic acid;

[0084] S2. Place the 96-well PCR plate containing the mouse tail lysis solution and mouse tail samples in a PCR instrument and incubate at 95 °C for 30 min;

[0085] S3. Use a multi-channel pipette to transfer the supernatant to a new PCR plate for storage;

[0086] S4. Use a multi-channel pipette to take the supernatant from the new PCR plate containing the supernatant as the PCR template and perform PCR amplification according to the following system and procedure;

[0087] The PCR amplification system consisted of 2 μl of the supernatant, 25 μl of 2x Taq Mix, and a primer set of 20 μmol / L. Sterile water was added to make the total volume of the reaction system 50 μl;

[0088] The PCR amplification reaction program was as follows: 95°C for 3 minutes; 95°C for 15 seconds, 60°C for 15 seconds, 72°C for 30 seconds, for 33 cycles; 72°C for 5 minutes.

[0089] S5. Take 10 μl of the obtained PCR product and run agarose gel electrophoresis.

[0090] Example group 5

[0091] S1. Take 48 1-2 mm mouse tail samples and clamp them into the wells of a 96-well PCR plate. Add 50 μl of mouse tail lysate to each well containing the mouse tail sample to cover the mouse tail sample. The components of the mouse tail lysate were 150 mmol / L sodium hydroxide, 4 mol / L isothiocyanate, 2 mol / L guanidine hydrochloride, 300 mmol / L sodium chloride, and 2 mol / L ethylenediaminetetraacetic acid;

[0092] S2. Place the 96-well PCR plate containing the mouse tail lysate and mouse tail samples in a PCR instrument and incubate at 95°C for 30 min;

[0093] S3. Use a multi-channel pipette to transfer the supernatant to a new PCR plate for storage;

[0094] S4. Use a multi-channel pipette to take the supernatant from the new PCR plate containing the supernatant as the PCR template and perform PCR amplification according to the following system and program;

[0095] The PCR amplification system consisted of 2 μl of the supernatant, 25 μl of 2x Taq Mix, and a primer set of 20 μmol / L. Sterile water was added to make the total volume of the reaction system 50 μl;

[0096] The PCR amplification reaction program was as follows: 95°C for 3 minutes; 95°C for 15 seconds, 60°C for 15 seconds, 72°C for 30 seconds, for 33 cycles; 72°C for 5 minutes.

[0097] S5. Take 10 μl of the obtained PCR product and run agarose gel electrophoresis.

[0098] The electrophoresis results of Example groups 1-4 were Figure 1 as shown. Example group 1 corresponded to Figure 1 A1 - A4 in Figure 1 Example group 2 corresponded to Figure 1 A5 - A8 in Figure 1 Example group 3 corresponded to B1 - B4 inFigure 2 shown.

[0099] The difference between Example Groups 1-4 is that the components of the rat tail lysate used are different. Example Groups 1-4 are all within the scope of the present invention. Figure 1 It can be seen that amplification bands can be seen in A1-A8 and B1-B8, proving that the mouse tail lysis solution can achieve lysis of mouse tail tissue with a good degree of lysis under simple operation. Although the mouse tail tissue used is small and no centrifugation purification operation is performed, PCR amplification can be directly performed through the supernatant after lysis, and the corresponding bands can be run out, with a success rate of 100%.

[0100] The rat tail lysate used in Example 4 was under the optimal content limit. Figure 1 It can be seen that the brightness of the bands produced by A1, A4, A7 and B2 is insufficient and the effect is uneven, but the bands of B5-B8 are not insufficient in brightness, indicating that the mouse tail lysate at the optimal content can achieve a 100% success rate and uniform results.

[0101] The results obtained in Example 5 are as follows Figure 2 As shown, Example Group 5 used a rat tail lysis buffer with an optimal component ratio to lyse 48 rat tail samples. The sample amplification bands after batch lysis were single and had normal brightness, which means that the rat tail lysis buffer can be used to batch lyse rat tail samples to obtain templates for subsequent PCR amplification, thereby achieving the purpose of high-throughput cell nucleic acid lysis.

[0102] The technical principle of the present invention is described above in conjunction with specific embodiments. These descriptions are only for explaining the principle of the present invention and cannot be interpreted as limiting the scope of protection of the present invention in any way. Based on the explanations herein, those skilled in the art can associate other specific implementations of the present invention without paying creative labor, and these methods will fall within the scope of protection of the present invention.

Claims

1. A rat tail lysate, characterized in that: The invention comprises the following components: 20-200 mmol / L of sodium hydroxide, 2-5 mol / L of isosulfate, 1.5-3 mol / L of guanidine hydrochloride, 100-500 mmol / L of sodium chloride and 1-3 mol / L of ethylenediaminetetraacetic acid.

2. The rat tail lysate according to claim 1, characterized in that The invention comprises the following components: 150 mmol / L sodium hydroxide, 4 mol / L isothioic acid ester, 2 mol / L guanidine hydrochloride, 300 mmol / L sodium chloride and 2 mol / L ethylenediaminetetraacetic acid.

3. A rat tail rapid PCR amplification sequencing detection method, characterized in that: The following steps are involved: S1. Take rat tail samples and place them in different wells of a PCR plate, and add the rat tail lysate described in any one of claims 1 to 2 to each well to soak the rat tail sample; S2, placing the PCR plate containing the rat tail lysis solution and the rat tail sample in a PCR instrument, heating and incubating until fully lysed; S3. Use a multichannel pipette to transfer the supernatant to a new PCR plate for storage; S4. Taking the supernatant for PCR amplification.

4. A rat tail rapid PCR amplification sequencing detection method according to claim 3, characterized in that: In step S1, the amount of the rat tail lysate added is 50-150 μl.

5. A rat tail rapid PCR amplification sequencing detection method according to claim 4, characterized in that: In step S1, the length of the rat tail sample is 1-2 mm.

6. A rat tail rapid PCR amplification sequencing detection method according to claim 3, characterized in that: In step S2, incubate at 95-100°C for 15-30 min.

7. A rat tail rapid PCR amplification sequencing detection method according to claim 3, characterized in that: In step S4, 2 μl of the supernatant is placed in a new PCR plate for PCR amplification.

8. A rat tail rapid PCR amplification sequencing detection method according to claim 3, characterized in that: In step S4, the PCR amplification system is 2 μl of supernatant, 25 μl of 2x Taq Mix and 20 μmol / L of primer set, and sterile water is added to make the total volume of the reaction system 50 μl; The reaction program of PCR amplification was as follows: 95°C for 3 minutes; 95°C for 15 seconds, 60°C for 15 seconds, 72°C for 30 seconds, 33 cycles; 72°C for 5 minutes.

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