Tubular incision enzyme premixed liquid and application thereof

Abstract

The invention relates to a tubular incision enzyme premixed solution and application thereof, and belongs to the technical field of biology. The invention provides a tubular endonuclease premixed solution. The tubular endonuclease premixed solution is prepared from a solvent, restriction endonuclease, potassium acetate, magnesium acetate, NaCL, KCL, Triton X-100, bovine serum albumin, dithiothreitol, glycerol and trehalose. The tubular endonuclease premixed solution has the functions of buffering and enzyme digestion, and when the tubular endonuclease premixed solution is used for enzyme digestion reaction, the tubular endonuclease premixed solution is mixed with a DNA substrate and then incubated, so that an enzyme digestion product can be recycled, and additional calculation and component addition are not needed. Moreover, when the tubular endonuclease premix liquid is used for enzyme digestion reaction, the tubular endonuclease premix liquid containing different restriction endonuclease can simultaneously perform enzyme digestion on the same enzyme digestion object, the compatibility is strong, and normal enzyme digestion can be performed on the enzyme digestion objects with different volumes and different masses.

Description

Technical Field

[0001] The invention relates to a one-tube endonuclease premix and application thereof, belonging to the field of biotechnology. Background Art

[0002] Restriction endonucleases (also called restriction endonucleases) can recognize and attach to specific deoxyribonucleotide sequences and cut the phosphodiester bond between two deoxyribonucleotides at specific locations in each chain. Restriction enzymes play an important role in genetic engineering and are currently widely used in fields such as molecular cloning (also known as DNA recombination). Different restriction endonucleases recognize different DNA sequences. They can cut DNA within the recognition sequence or at a position not far from the recognition sequence, and after enzyme cutting, sticky ends or blunt ends are formed.

[0003] According to the structure of restriction enzymes, the need for cofactors and the mode of action, restriction enzymes can be divided into four types: type I, type II, type III and type IV. Among them, type I restriction endonucleases cut at a position far away from the recognition sequence, and have both recognition and cutting and modification activities; type II restriction endonucleases cut within or at a specific short distance from the recognition sequence, and only have the function of recognition and cutting; type III restriction endonucleases cut at a position 25 to 27 bp away from the recognition sequence, and have both recognition and cutting and modification activities; type IV restriction endonucleases cut at a position 28 to 35 bp away from the recognition sequence, and only cut methylated DNA sequences. At present, most commercial endonucleases are type II restriction endonucleases.

[0004] Molecular cloning technology is a method of purifying and amplifying specific DNA fragments at the molecular level. In the process of molecular cloning, the target gene needs to be inserted into a cloning vector to form a recombinant cloning vector, and then the recombinant cloning vector is introduced into a suitable host for replication and amplification of the target gene. Among them, the cloning vector used in molecular cloning technology generally refers to a plasmid, and the plasmid usually needs to be linearized before it can be connected to the target fragment. At this stage, restriction enzymes are the most commonly used tool enzymes to obtain linearized plasmids. When using restriction enzymes to obtain linearized plasmids, the corresponding enzyme digestion reaction system must also be prepared. At present, the most common enzyme digestion reaction system mainly includes four components: 10× enzyme digestion buffer, substrate DNA (when obtaining linearized plasmids, the plasmid itself is the substrate DNA), restriction endonuclease working solution and water.

[0005] However, this enzyme digestion reaction system still has many defects. For example, this enzyme digestion reaction system has a fixed reaction volume, generally 1 μg of plasmid DNA is digested in a 50 μL enzyme digestion reaction system, and the substrate input volume and the volume of water added need to be calculated according to the concentration of the substrate during preparation, which is easy to cause calculation errors or confusion, leading to experimental errors; when preparing this enzyme digestion reaction system, each component needs to be added to the reaction tube separately in sequence, and the components are more and the volume is smaller, which is not only easy to cause aspiration errors, but also complicated to operate, greatly increasing the experimental time; the amount of restriction endonuclease working solution in this enzyme digestion reaction system is extremely low (generally about 1 μL), while the glycerol content of the restriction endonuclease working solution is extremely high (generally about 50%), and the glycerol is viscous, which makes it easier to cause aspiration errors when aspirating the restriction endonuclease working solution to prepare the enzyme digestion reaction system. The enzyme digestion reaction system is poor in quality; and, in order to maintain stability, the enzyme digestion reaction system needs to be stored in separate tubes, generally in two tubes, one tube storing the restriction endonuclease working solution, and the other tube storing the 10× enzyme digestion buffer, and the packaging cost is high; in addition, after the plasmid is linearized using the enzyme digestion reaction system, an agarose gel recovery kit is required to recover the enzyme digestion product, but the agarose gel recovery kit is only suitable for the recovery of a small amount of enzyme digestion product (the recovery amount generally does not exceed 10 μg) and the recovery efficiency is low (the recovery efficiency is generally only 30-40%). If a large number of linearized plasmids need to be prepared, a multi-tube enzyme digestion reaction system needs to be prepared to linearize the plasmids separately and then recover them. This not only makes the enzyme digestion process cumbersome, but also increases the workload and time of subsequent recovery, and is not suitable for large-scale continuous enzyme digestion to recover plasmids. Summary of the invention

[0006] To solve the above problems, the present invention provides a one-tube endonuclease premix solution, the components of which include a solvent, a restriction endonuclease, potassium acetate, magnesium acetate, NaCL, KCL, Triton X-100, bovine serum albumin (BSA), dithiothreitol (DTT), glycerol and trehalose.

[0007] In one embodiment of the present invention, the concentration of the restriction endonuclease in the solvent is 0.5-2U / μL; the concentration of the potassium acetate in the solvent is 30-50mM; the concentration of the magnesium acetate in the solvent is 5-10mM; the concentration of the NaCL in the solvent is 20-200mM; the concentration of the KCL in the solvent is 2-5mM; in terms of volume percentage, the concentration of Triton X-100 in the solvent is 0.05-0.15%; the concentration of the bovine serum albumin in the solvent is 120-200μg / mL; the concentration of the dithiothreitol in the solvent is 0.1-1mM; in terms of volume percentage, the concentration of the glycerol in the solvent is 5-20%; in terms of mass volume percentage (w / v, g / 100mL), the concentration of the trehalose in the solvent is 2-5%.

[0008] In one embodiment of the present invention, the concentration of the restriction endonuclease in the solvent is 2U / μL; the concentration of the potassium acetate in the solvent is 50mM; the concentration of the magnesium acetate in the solvent is 10mM; the concentration of the NaCL in the solvent is 30mM; the concentration of the KCL in the solvent is 2mM; in terms of volume percentage, the concentration of Triton X-100 in the solvent is 0.1%; the concentration of the bovine serum albumin in the solvent is 200μg / mL; the concentration of the dithiothreitol in the solvent is 0.1mM; in terms of volume percentage, the concentration of the glycerol in the solvent is 3%; in terms of mass volume percentage (w / v, g / 100mL), the concentration of the trehalose in the solvent is 5%.

[0009] In one embodiment of the present invention, the solvent comprises a buffer; the buffer comprises a Tirs-CH3COOH buffer (Tris-acetate buffer) and / or a Tris-HCL buffer (Tris-hydrochloric acid buffer).

[0010] In one embodiment of the present invention, the solvent is Tirs-CH3COOH buffer; the concentration of the Tirs-CH3COOH buffer is 10-30 mM and the pH is 7.4-8.0.

[0011] In one embodiment of the present invention, the concentration of the Tirs-CH3COOH buffer is 30 mM and the pH is 7.9.

[0012] In one embodiment of the present invention, the components of the one-tube endonuclease premix solution are composed of a solvent, a restriction endonuclease, potassium acetate, magnesium acetate, NaCL, KCL, Triton X-100, bovine serum albumin (BSA), dithiothreitol (DTT), glycerol and trehalose.

[0013] In one embodiment of the present invention, the restriction endonuclease is a Type II restriction endonuclease.

[0014] The present invention also provides an enzyme cleavage reaction system, which comprises the above-mentioned one-tube endonuclease premix and an enzyme cleavage object.

[0015] In one embodiment of the present invention, in the enzyme cleavage reaction system, the ratio of a one-tube endonuclease premix to the enzyme cleavage object is 1 to 5 μg: 10 μL (a one-tube endonuclease premix contains only one restriction endonuclease. If the same enzyme cleavage reaction system uses more than two one-tube endonuclease premixes to perform enzyme cleavage on the same enzyme cleavage object, then in the enzyme cleavage reaction system, the ratio of each one-tube endonuclease premix to the enzyme cleavage object is 1 to 5 μg: 10 μL).

[0016] In one embodiment of the present invention, the enzyme cutting object includes double-stranded DNA; the double-stranded DNA includes plasmid, PCR product, genome and / or DNA fragment.

[0017] The present invention also provides an enzyme cutting method, which comprises: using the above-mentioned one-tube endonuclease premix to perform enzyme cutting on the enzyme cutting object, and then recovering the enzyme cutting product or performing electrophoresis detection on the enzyme cutting product.

[0018] In one embodiment of the present invention, the enzyme cleavage method comprises: mixing the above-mentioned one-tube endonuclease premix and the enzyme cleavage object to obtain an enzyme cleavage reaction system; after incubating the enzyme cleavage reaction system, recovering the enzyme cleavage product or performing electrophoresis detection on the enzyme cleavage product.

[0019] In one embodiment of the present invention, the enzymatic cleavage method comprises: mixing the above-mentioned tubular endonuclease premix and the enzymatic cleavage object to obtain an enzymatic cleavage reaction system; incubating the enzymatic cleavage reaction system to obtain an incubation product; using a liquid input device to input the incubation product into an ultrafiltration device for ultrafiltration, and then recovering the enzymatic cleavage product; the ultrafiltration device can be passed by the restriction endonuclease, but cannot be passed by the enzymatic cleavage product (during the incubation and input process, the restriction endonuclease in the tubular endonuclease premix plays an enzymatic cleavage role to cut the enzymatic cleavage object; during the ultrafiltration process, since the restriction endonuclease is a protein, it can pass through the ultrafiltration device, while the enzymatic cleavage product cannot pass through the ultrafiltration device, so the restriction endonuclease can be recovered and reused through the output end of the ultrafiltration device, and the enzymatic cleavage product can be concentrated and recovered through the reflux end of the ultrafiltration device).

[0020] In one embodiment of the present invention, the liquid input device is a peristaltic pump; the ultrafiltration device is an ultrafiltration membrane package; the ultrafiltration membrane package has a certain retention pore size, so that it can be passed through by restriction endonucleases but not by enzyme cleavage products.

[0021] In one embodiment of the present invention, the mixing ratio of the one-tube endonuclease premix and the enzyme cleavage object is 1-5 μg:10 μL (a one-tube endonuclease premix contains only one restriction endonuclease. If the same enzyme cleavage reaction system uses more than two one-tube endonuclease premixes to perform enzyme cleavage on the same enzyme cleavage object, then in the enzyme cleavage reaction system, the ratio of each one-tube endonuclease premix and the enzyme cleavage object is 1-5 μg:10 μL).

[0022] In one embodiment of the present invention, the incubation temperature is 30-37° C. and the incubation time is 5-30 min.

[0023] In one embodiment of the present invention, the input flow rate of the incubation product in the ultrafiltration device is 5 to 30 mL / min.

[0024] In one embodiment of the present invention, the molecular weight cutoff of the filter membrane is 2 to 5 times the molecular weight of the restriction endonuclease.

[0025] In one embodiment of the present invention, the molecular weight cutoff of the filter membrane is 3 times the molecular weight of the restriction endonuclease.

[0026] In one embodiment of the present invention, the material of the filter membrane is polyethersulfone.

[0027] In one embodiment of the present invention, the enzyme cutting object includes double-stranded DNA; the double-stranded DNA includes plasmid, PCR product, genome and / or DNA fragment.

[0028] In one embodiment of the present invention, when the object of enzyme cleavage is a plasmid, the enzyme cleavage product is a linearized plasmid.

[0029] The present invention also provides the use of the one-tube endonuclease premix solution or the enzyme digestion reaction system or the enzyme digestion method in enzyme digestion.

[0030] In one embodiment of the present invention, the enzyme digestion includes plasmid linearization, gene library construction and / or restriction fragment length polymorphism (RFLP) analysis by enzyme digestion.

[0031] The technical solution of the present invention has the following advantages:

[0032] The present invention provides a one-tube endonuclease premix solution, wherein the components of the one-tube endonuclease premix solution include a solvent, a restriction endonuclease, potassium acetate, magnesium acetate, NaCL, KCL, Triton X-100, bovine serum albumin (BSA), dithiothreitol (DTT), glycerol and trehalose. The one-tube endonuclease premix solution has the following advantages:

[0033] First, the one-tube endonuclease premix has both buffering and enzyme cleavage functions. When the one-tube endonuclease premix is ​​used for enzyme cleavage reaction, it is only necessary to mix the one-tube endonuclease premix with the DNA substrate and then incubate to recover the enzyme cleavage product without additional calculation and component addition, which has the advantages of small error, simple operation and low time cost;

[0034] Second, when the one-tube endonuclease premix is ​​used for enzyme digestion reaction, the one-tube endonuclease premix containing different restriction endonucleases can digest the same digestion object at the same time, has strong compatibility, can digest digestion objects of different volumes and masses normally, shortens the digestion time, and is easy to apply to large-scale digestion reactions;

[0035] Third, the one-tube endonuclease premix solution has the advantage of high stability in that the enzyme cleavage performance remains basically unchanged after long-term storage under different storage conditions (4°C, -20°C, and repeated freezing and thawing, etc.);

[0036] Fourthly, the one-tube endonuclease premix does not need to be stored in separate tubes, and the glycerol content of the one-tube endonuclease premix is ​​not high, which has the advantages of low cost and easy absorption;

[0037] Fifth, after the one-tube endonuclease premix is ​​used for the enzyme digestion reaction, agarose gel electrophoresis is not required, and the digestion product can be recovered only by ultrafiltration, which reduces the operation steps, saves time cost, and is easy to be applied to large-scale continuous enzyme digestion to recover plasmids. At the same time, the ultrafiltration process realizes the reuse of restriction endonucleases, saving reagent costs.

[0038] Therefore, the one-tube endonuclease premix has great application prospects in the field of enzyme digestion, and can achieve large-scale continuous enzyme digestion and recovery of enzyme digestion objects such as plasmids. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 :The optimal solution for premix was used to screen the results of enzyme electrophoresis.

[0040] Figure 2 :One-tube endonuclease premix stability verification enzyme electrophoresis results.

[0041] Figure 3 :One-tube endonuclease premix solution stability verification of enzyme digestion and conversion results.

[0042] Figure 4 :One tube endonuclease premix solution is stored at -20℃ for 60 days to verify the stability of enzyme electrophoresis results.

[0043] Figure 5 :A tube of endonuclease premix is ​​stored at -20℃ for 60 days to verify the stability of the enzyme digestion and conversion results.

[0044] Figure 6 :One-tube endonuclease premix substrate compatibility verification enzyme electrophoresis results.

[0045] Figure 7 : Results of large-volume plasmid electrophoresis using one-tube endonuclease premix.

[0046] Figure 8 : One-tube endonuclease premix solution large volume enzyme digestion and plasmid electrophoresis results. DETAILED DESCRIPTION

[0047] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0048] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0049] Example 1 to Example 6: A one-tube endonuclease premix

[0050] The present embodiment provides a one-tube endonuclease premix solution, the components of the one-tube endonuclease premix solution are Tirs-CH3COOH buffer, restriction endonuclease, potassium acetate, magnesium acetate, NaCL, KCL, Triton X-100, bovine serum albumin (BSA), dithiothreitol (DTT), glycerol and trehalose, and the specific formula is shown in Table 1.

[0051] Table 1: Formula of one-tube endonuclease premix

[0052]

[0053]

[0054] Comparative Example 1: A reagent for enzyme digestion

[0055] This comparative example provides an enzyme cutting reagent, which consists of 10× enzyme cutting buffer, restriction endonuclease working solution and water. The formula of 10× enzyme cutting buffer is specifically shown in Table 2, and the formula of restriction endonuclease working solution is specifically shown in Table 3.

[0056] Table 2 10× enzyme digestion buffer recipe

[0057]

[0058] Table 3 Restriction endonuclease working solution

[0059] Element concentration Endonuclease 20 U / μL Tris-HCl buffer (pH 7.4) 10mM NaCl 100mM Ethylenediaminetetraacetic acid (EDTA) 1mM Bovine serum albumin (BSA) 200 μg / mL Glycerol (v / v) 50％

[0060] Experimental Example 1: Enzyme Digestion Performance Verification of One-Tube Endonuclease Premix

[0061] In this experimental example, pGADT7 plasmid (purchased from Qingke Biotechnology) was used as the restriction endonuclease 1, NcoI (purchased from Qingke Biotechnology) was used as the restriction endonuclease 1, EcoRV (purchased from Qingke Biotechnology) was used as the restriction endonuclease 2, and the enzyme cutting reagent of Comparative Example 1 was used as a control. The one-tube endonuclease premix in Examples 1 to 6 was used to perform enzyme cutting on the enzyme cutting object to verify its enzyme cutting performance. The specific process is as follows:

[0062] Experimental group 1: 1 μg of the digestion object was mixed with 10 μL of different premixes (premix 1, premix 2, premix 3, premix 4, premix 5 or premix 6) containing restriction endonuclease 1 to obtain a single digestion system; the single digestion system was first incubated at 37°C for 15 min and then incubated at 80°C for 20 min.

[0063] Experimental group 2: 1 μg of the digestion object was mixed with 10 μL of different premixes containing restriction endonuclease 2 (premix 1, premix 2, premix 3, premix 4, premix 5 or premix 6) to obtain a single digestion system; the single digestion system was first incubated at 37°C for 15 min and then incubated at 80°C for 20 min.

[0064] Experimental group 3: 1 μg of the digestion object was mixed with 10 μL of different premixes containing restriction endonuclease 1 (premix 1, premix 2, premix 3, premix 4, premix 5 or premix 6) and 10 μL of different premixes containing restriction endonuclease 2 (premix 1, premix 2, premix 3, premix 4, premix 5 or premix 6) to obtain a double digestion system; the double digestion system was first incubated at 37°C for 15 min and then incubated at 80°C for 20 min.

[0065] Control group 1: 1 μg of the digestion object and 5 μL of 10× digestion buffer were mixed, and water was added to make up the total volume to 50 μL to obtain a plasmid blank control system; the plasmid blank control system was first incubated at 37°C for 15 min, and then incubated at 80°C for 20 min.

[0066] Control group 2: 1 μg of the digestion object, 5 μL of 10× digestion buffer and 1 μL of restriction endonuclease working solution containing restriction endonuclease 1 were mixed, and water was added to make the total volume 50 μL to obtain a single digestion system; the single digestion system was first incubated at 37°C for 15 min and then incubated at 80°C for 20 min.

[0067] Control group 3: 1 μg of the digestion object, 5 μL of 10× digestion buffer and 1 μL of restriction endonuclease working solution containing restriction endonuclease 2 were mixed, and water was added to make the total volume 50 μL to obtain a single digestion system; the single digestion system was first incubated at 37°C for 15 min and then incubated at 80°C for 20 min.

[0068] Control group 4: 1 μg of the digestion object, 5 μL of 10× digestion buffer, 1 μL of restriction endonuclease working solution containing restriction endonuclease 1 and 1 μL of restriction endonuclease working solution containing restriction endonuclease 2 were mixed, and water was added to make the total volume 50 μL to obtain a double digestion system; the double digestion system was first incubated at 37°C for 15 min and then incubated at 80°C for 20 min.

[0069] After the incubation, the enzyme digestion products of each group were detected by agarose gel electrophoresis. The detection results are shown in Figure 1 .according to Figure 1 The results show that the one-tube endonuclease premix 1 to one-tube endonuclease premix 6 in Examples 1 to 6 can all normally digest the plasmid. Compared with the digestion reagent of Comparative Example 1, the digestion products obtained by premix 1 to premix 5 have obvious dragging, while premix 6 has no obvious dragging, and the digestion effect is better.

[0070] Experimental Example 2: Stability Verification of One-Tube Endonuclease Premix

[0071] In this experimental example, pGADT7 plasmid was used as the digestion object, and the digestion reagent of comparative example 1 was used as the control. The one-tube endonuclease premix 6 in Example 6 was stored for a long time under different storage conditions (4°C, -20°C, and repeated freezing and thawing, etc.) and then digested the digestion object to verify its stability. The specific process is as follows:

[0072] Repeated freeze-thaw group:

[0073] The premixed solutions 6 containing restriction endonuclease NdeI, restriction endonuclease NheI, restriction endonuclease NotI, restriction endonuclease EcoRI, restriction endonuclease PvuII, restriction endonuclease BamHI, restriction endonuclease EcoRV or restriction endonuclease NcoI were first frozen at -20°C for 60 minutes and then thawed on ice. The premixed solutions 6 containing different restriction endonucleases were repeatedly frozen and thawed for 20 cycles, with one freezing and one thawing process as one cycle, to obtain premixed solutions 6 containing different restriction endonucleases that were repeatedly frozen and thawed.

[0074] The digestion target was digested using a premix 6 containing different restriction endonucleases that had been repeatedly frozen and thawed. The digestion scheme was as follows:

[0075] Experimental group 1: 1 μg of the digestion object was mixed with 10 μL of premix 6 containing restriction endonuclease NdeI and 10 μL of premix 6 containing restriction endonuclease NheI to obtain a double digestion system; the double digestion system was first incubated at 37°C for 15 min and then incubated at 80°C for 20 min.

[0076] Experimental group 2: 1 μg of the digestion object was mixed with 10 μL of premix 6 containing restriction endonuclease NotI and 10 μL of premix 6 containing restriction endonuclease EcoRI to obtain a double digestion system; the double digestion system was first incubated at 37°C for 15 min and then incubated at 80°C for 20 min.

[0077] Experimental group 3: 1 μg of the digestion object was mixed with 10 μL of premix 6 containing restriction endonuclease PvuII and 10 μL of premix 6 containing restriction endonuclease BamHI to obtain a double digestion system; the double digestion system was first incubated at 37°C for 15 min and then incubated at 80°C for 20 min.

[0078] Experimental group 4: 1 μg of the digestion object was mixed with 10 μL of premix 6 containing restriction endonuclease EcoRV and 10 μL of premix 6 containing restriction endonuclease NcoI to obtain a double digestion system; the double digestion system was first incubated at 37°C for 15 min and then incubated at 80°C for 20 min.

[0079] Control group 1: 1 μg of the digestion object, 5 μL of 10× digestion buffer, 1 μL of a restriction endonuclease working solution containing a restriction endonuclease NdeI, and 1 μL of a restriction endonuclease working solution containing a restriction endonuclease NheI were mixed, and water was added to make the total volume 50 μL to obtain a double digestion system; the double digestion system was first incubated at 37°C for 15 min and then incubated at 80°C for 20 min.

[0080] Control group 2: 1 μg of the digestion object, 5 μL of 10× digestion buffer, 1 μL of a restriction endonuclease working solution containing a restriction endonuclease NotI, and 1 μL of a restriction endonuclease working solution containing a restriction endonuclease EcoRI were mixed, and water was added to make the total volume 50 μL to obtain a double digestion system; the double digestion system was first incubated at 37°C for 15 min, and then incubated at 80°C for 20 min.

[0081] Control group 3: 1 μg of the digestion object, 5 μL of 10× digestion buffer, 1 μL of restriction endonuclease working solution containing restriction endonuclease PvulI and 1 μL of restriction endonuclease working solution containing restriction endonuclease BamHI were mixed, and water was added to make the total volume 50 μL to obtain a double digestion system; the double digestion system was first incubated at 37°C for 15 min and then incubated at 80°C for 20 min.

[0082] Control group 4: 1 μg of the digestion object, 5 μL of 10× digestion buffer, 1 μL of restriction endonuclease working solution containing restriction endonuclease EcoRV and 1 μL of restriction endonuclease working solution containing restriction endonuclease NcoI were mixed, and water was added to make the total volume 50 μL to obtain a double digestion system; the double digestion system was first incubated at 37°C for 15 min and then incubated at 80°C for 20 min.

[0083] After the incubation, the enzyme digestion products of each group were detected by agarose gel electrophoresis. The detection results are shown in Figure 2 The enzymatic cleavage efficiency of each group of enzyme products was tested through transformation experiments. The test results are shown in Figure 3 and Table 4; wherein, the transformation experiment process is as follows: 5 μL of the obtained enzyme digestion product of each group is added to 100 μL of Escherichia coli DH5α chemical transformation competent cells (purchased from Qingke Biotechnology), and then heat-shocked at 42°C for 60s to obtain a transformation product; 700 μL of antibiotic-free LB liquid culture medium (purchased from Qingke Biotechnology) is added to the transformation product, and the product is resuscitated and cultured in a shaker at 37°C and 220rpm for 30min to obtain a culture solution; 200 μL of the culture solution is spread on an LB solid plate (purchased from Qingke Biotechnology) containing ampicillin (working concentration 100 μg / mL), and after the coating is completed, the plate is inverted and cultured in a 37°C constant temperature incubator for 16h. After the culture is completed, the transformants are counted and the enzyme digestion efficiency of each group is calculated.

[0084] 4℃ storage group:

[0085] The premix 6 containing restriction endonuclease NdeI, restriction endonuclease NheI, restriction endonuclease NotI, restriction endonuclease EcoRI, restriction endonuclease PvuII, restriction endonuclease BamHI, restriction endonuclease EcoRV or restriction endonuclease NcoI was stored at 4°C for 30 days, and then stability was verified according to the method of the repeated freezing and thawing group. The verification results are shown in Figure 2-Figure 3 and Table 4.

[0086] -20℃ storage for 30 days group:

[0087] The premix 6 containing restriction endonuclease NdeI, restriction endonuclease NheI, restriction endonuclease NotI, restriction endonuclease EcoRI, restriction endonuclease PvuII, restriction endonuclease BamHI, restriction endonuclease EcoRV or restriction endonuclease NcoI was stored at -20°C for 30 days, and then stability was verified according to the method of the repeated freezing and thawing group. The verification results are shown in Figure 2-Figure 3 and Table 4.

[0088] -20℃ storage for 60 days:

[0089] The premixed solutions 6 containing restriction endonuclease NdeI, restriction endonuclease NheI, restriction endonuclease NotI, restriction endonuclease EcoRI, restriction endonuclease PvuII, restriction endonuclease BamHI, restriction endonuclease EcoRV or restriction endonuclease NcoI were stored at -20°C for 60 days to obtain premixed solutions 6 containing different restriction endonucleases that were stored at ultra-low temperatures for a long time.

[0090] The digestion target was digested using a premix 6 containing different restriction endonucleases that had been stored at ultra-low temperature for a long time. The digestion scheme was as follows:

[0091] Experimental group 1: 1 μg of the digestion object was mixed with 10 μL of premix 6 containing restriction endonuclease NdeI and 10 μL of premix 6 containing restriction endonuclease NheI to obtain a double digestion system; the double digestion system was first incubated at 37°C for 15 min and then incubated at 80°C for 20 min.

[0092] Experimental group 2: 1 μg of the digestion object was mixed with 10 μL of premix 6 containing restriction endonuclease SmaI and 10 μL of premix 6 containing restriction endonuclease PvuII to obtain a double digestion system; the double digestion system was first incubated at 37°C for 15 min and then incubated at 80°C for 20 min.

[0093] Experimental group 3: 1 μg of the digestion object was mixed with 10 μL of premix 6 containing restriction endonuclease BamHI and 10 μL of premix 6 containing restriction endonuclease NotI to obtain a double digestion system; the double digestion system was first incubated at 37°C for 15 min and then incubated at 80°C for 20 min.

[0094] Control group 1: 1 μg of the digestion target and 5 μL of 10× digestion buffer were mixed, and water was added to make up the total volume to 50 μL to obtain a plasmid control system; the plasmid control system was first incubated at 37°C for 15 min, and then incubated at 80°C for 20 min.

[0095] After the incubation, the enzyme digestion products of each group were detected by agarose gel electrophoresis. The detection results are shown in Figure 4 The enzymatic cleavage efficiency of each group of enzyme products was tested through transformation experiments. The test results are shown in Figure 5 and Table 5.

[0096] Depend on Figure 2 to Figure 5 As shown in Tables 4 and 5, although the content of glycerol and protein stabilizer in the one-tube endonuclease premix in Example 6 is very low, the enzymatic cleavage effect does not change significantly after being stored at 4°C for one month, -20°C for one month, -20°C for two months, and repeatedly frozen and thawed 20 times. This result shows that it has strong stability.

[0097] Table 4 Enzyme cleavage efficiency of the enzyme cleavage products obtained in each group

[0098]

[0099] Table 5 Enzyme cleavage efficiency of the enzyme cleavage products obtained in each group

[0100]

[0101] Experimental Example 3: Compatibility Verification of One-Tube Endonuclease Premix

[0102] In this experimental example, pGADT7 plasmid, pUC19 plasmid and pET28a plasmid (purchased from Qingke Biotechnology) were used as the digestion objects, and the digestion reagent of Comparative Example 1 was used as a control. The one-tube endonuclease premix 6 in Example 6 was used to digest the digestion objects of different volumes and masses to verify their compatibility. The specific process is as follows:

[0103] Experimental group: According to Table 6, 1 μg, 3 μg, and 5 μg of the digestion object were mixed with 10 μL of premix 6 containing restriction endonuclease to obtain a digestion system; the digestion system was first incubated at 37°C for 15 min and then incubated at 80°C for 20 min.

[0104] Control group: 1 μg of the digestion object and 5 μL of 10× digestion buffer were mixed, and water was added to make up the total volume to 50 μL to obtain a plasmid control system; the plasmid control system was first incubated at 37°C for 15 min, and then incubated at 80°C for 20 min.

[0105] After the incubation, the enzyme digestion products of each group were detected by agarose gel electrophoresis. The detection results are shown in Figure 6 .Depend on Figure 6 It can be seen that the one-tube endonuclease premix in Example 6 is well compatible with substrate DNA of different volumes and masses, and has a better enzyme digestion effect.

[0106] Table 6 Reaction system of one-tube endonuclease premix

[0107]

[0108]

[0109] Example 7: A method for recovering plasmids by large-volume enzyme digestion

[0110] This example provides a method for recovering plasmids by enzyme digestion of large volumes, and the method is as follows:

[0111] Using the one-tube endonuclease premix 6 in Example 6, the digestion object is mixed with the premix 6 containing restriction endonuclease 1 and the premix 6 containing restriction endonuclease 2 in a ratio of 1 μg: 10 μL: 10 μL to obtain a double digestion system; the double digestion system is incubated at 37° C. for 15 minutes to obtain an incubation product;

[0112] An ultrafiltration membrane package (pall, Minimate ultrafiltration membrane package, 100 kD) was connected to the pipeline of a peristaltic pump (Raffer, BT101s) to obtain an enzyme cleavage recovery system; the enzyme cleavage recovery system was first placed in a 37°C environment for temperature balance, and then the system was flushed with ultrapure water for 10 minutes (flushing the pipeline and the filter membrane), and then the ultrapure water in the system was drained to obtain a pretreated enzyme cleavage recovery system;

[0113] The incubation product is added to a beaker, and the peristaltic pump is turned on. The incubation product in the beaker is passed through a pipeline into an enzyme cleavage recovery system at a flow rate of 20 mL / min for ultrafiltration to recover the enzyme cleavage product. During the ultrafiltration process, the permeate flowing out of the output end of the enzyme cleavage recovery system is collected (the permeate contains restriction endonuclease and can be used for the next enzyme cleavage reaction), and the reflux end of the enzyme cleavage recovery system is connected to the beaker to which the incubation product is added, so that the enzyme cleavage product continues to be cleaved, concentrated and recovered, thereby ensuring that the enzyme cleavage object is completely cleaved and reaches an appropriate concentration.

[0114] Experimental Example 4: Feasibility verification of large-volume enzyme digestion and plasmid recovery method

[0115] In this experimental example, pGBKT7 plasmid (purchased from Qingke Biotechnology) was used as the restriction endonuclease object, NcoI was used as the restriction endonuclease 1, and AvrII was used as the restriction endonuclease 2. The large-volume restriction digestion and plasmid recovery method of Example 3 was used to digest and recover the restriction object to verify its feasibility. The specific process is as follows:

[0116] Experimental Group 1: According to the large-volume enzymatic digestion and plasmid recovery method of Example 3, 200 mL of the double enzymatic digestion system was digested and recovered. After all samples were concentrated to 50 mL, the reflux end of the enzymatic digestion recovery system was connected to a beaker to collect the linearized plasmid. The digestion products obtained by sampling after digestion and the collected linearized plasmid were detected by agarose gel electrophoresis, and the recovery rate of the collected linearized plasmid was calculated. The test results are shown in Figure 7-Figure 8 , the calculation results are shown in Table 7.

[0117] Experimental Group 2: According to the large-volume enzymatic digestion and plasmid recovery method of Example 3, 100 mL of the double enzymatic digestion system was digested and recovered. After all samples were concentrated to 30 mL, the reflux end of the enzymatic digestion recovery system was connected to a beaker to collect the linearized plasmid. The digestion products obtained by sampling after digestion and the collected linearized plasmid were detected by agarose gel electrophoresis, and the recovery rate of the collected linearized plasmid was calculated. The test results are shown in Figure 7-Figure 8 , the calculation results are shown in Table 7.

[0118] Control group: Use the enzyme digestion reagent of comparative example 1, mix 8 μg of the digestion object, 40 μL of 10× digestion buffer, 8 μL of restriction endonuclease working solution containing restriction endonuclease 1 and 8 μL of restriction endonuclease working solution containing restriction endonuclease 2, add water to make the total volume 400 μL, and obtain a double digestion system; incubate the double digestion system at 37°C for 15 minutes, and then incubate at 80°C for 20 minutes; use a gel recovery kit (purchased from Qingke Bio) to collect the linearized plasmid. The digestion products obtained by sampling after digestion and the collected linearized plasmid were detected by agarose gel electrophoresis, and the recovery rate of the collected linearized plasmid was calculated. The test results are shown in Figure 7-Figure 8 , the calculation results are shown in Table 7.

[0119] Depend on Figure 7-Figure 8 As shown in Table 7, the electrophoresis results of the linearized plasmid obtained by enzymatic digestion and recovery of the enzymatic digestion object using the large-volume enzymatic digestion and plasmid recovery method of Example 3 are consistent with those of the control group. According to the recovery rate results, it can be seen that the recovery rate of the enzymatic digestion method of Example 3 is higher than that of the gel recovery kit used in the control group.

[0120] Table 7 Recovery rate of the collected linearized plasmids

[0121]

[0122]

[0123] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A one-tube endonuclease premix solution, characterized in that: The components of the one-tube endonuclease premix solution include solvent, restriction endonuclease, potassium acetate, magnesium acetate, NaCL, KCL, Triton X-100, bovine serum albumin, dithiothreitol, glycerol and trehalose.

2. The one-tube endonuclease premix according to claim 1, characterized in that: The concentration of the restriction endonuclease in the solvent is 0.5-2U / μL; the concentration of the potassium acetate in the solvent is 30-50mM; the concentration of the magnesium acetate in the solvent is 5-10mM; the concentration of the NaCL in the solvent is 20-100mM; the concentration of the KCL in the solvent is 2-5mM; in terms of volume percentage, the concentration of Triton X-100 in the solvent is 0.05-0.15%; the concentration of the bovine serum albumin in the solvent is 120-200μg / mL; the concentration of the dithiothreitol in the solvent is 0.1-1mM; in terms of volume percentage, the concentration of the glycerol in the solvent is 5-20%; and in terms of mass volume percentage, the concentration of the trehalose in the solvent is 2-5%.

3. The one-tube endonuclease premix according to claim 1 or 2, characterized in that: The concentration of the restriction endonuclease in the solvent is 2U / μL; the concentration of the potassium acetate in the solvent is 50mM; the concentration of the magnesium acetate in the solvent is 10mM; the concentration of the NaCL in the solvent is 30mM; the concentration of the KCL in the solvent is 2mM; in terms of volume percentage, the concentration of Triton X-100 in the solvent is 0.1%; the concentration of the bovine serum albumin in the solvent is 200μg / mL; the concentration of the dithiothreitol in the solvent is 0.1mM; in terms of volume percentage, the concentration of the glycerol in the solvent is 3%; and in terms of mass volume percentage, the concentration of the trehalose in the solvent is 5%.

4. The one-tube endonuclease premix according to any one of claims 1 to 3, characterized in that: The solvent comprises a buffer; the buffer comprises a Tirs-CH3COOH buffer and / or a Tris-HCL buffer.

5. The one-tube endonuclease premix according to claim 4, characterized in that: The solvent is Tirs-CH3COOH buffer; the concentration of the Tirs-CH3COOH buffer is 10-30 mM and the pH is 7.4-8.

0.

6. An enzyme digestion reaction system, characterized in that: The enzyme digestion reaction system comprises the one-tube endonuclease premix solution according to any one of claims 1 to 5 and an enzyme digestion object.

7. An enzyme digestion method, characterized in that: The enzyme digestion method comprises: using the one-tube endonuclease premix solution according to any one of claims 1 to 5 to digest the digestion object, and then recovering the digestion product or performing electrophoresis detection on the digestion product.

8. The enzymatic digestion method according to claim 7, characterized in that: The enzyme digestion method comprises: mixing the one-tube endonuclease premix according to any one of claims 1 to 5 with an enzyme digestion object to obtain an enzyme digestion reaction system; after incubating the enzyme digestion reaction system, recovering the enzyme digestion product or performing electrophoresis detection on the enzyme digestion product.

9. The enzyme digestion method according to claim 7 or 8, characterized in that: The enzyme cleavage method comprises: mixing a tubular endonuclease premix according to any one of claims 1 to 6 with an enzyme cleavage object to obtain an enzyme cleavage reaction system; incubating the enzyme cleavage reaction system to obtain an incubation product; using a liquid input device to input the incubation product into an ultrafiltration device for ultrafiltration, and then recovering the enzyme cleavage product; the ultrafiltration device can be passed by the restriction endonuclease, but cannot be passed by the enzyme cleavage product.

10. Use of the one-tube endonuclease premix solution according to any one of claims 1 to 5, the enzyme digestion reaction system according to claim 6, or the enzyme digestion method according to any one of claims 7 to 9 in enzyme digestion.

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