A high-concentration plasmid extraction method based on a kit
By optimizing the operation steps of the Megi Biomass Grain Rapid Retrieval Kit, the problems of low plasmid concentration and high cost in the existing plasmid extraction methods are solved, and efficient and low-cost high-concentration plasmid DNA extraction is achieved, meeting the needs of protoplast transformation experiments.
Patent Information
- Application Number
- CN202411920278.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-25
- Publication Date
- 2025-04-04
AI Technical Summary
The plasmid concentration extracted by the existing plasmid extraction kit is low, which is difficult to meet the high requirements for plasmid concentration and purity in protoplast transformation experiments. Moreover, the traditional plasmid purification method takes a long time and is costly.
Based on Megi Bio's plasmid rapid extraction kit, optimized operation steps, including increasing the volume of bacterial fluid, using a larger centrifuge tube, increasing the amount of buffer, increasing the number of columns, and using multiple accumulated washings to extract and purify high concentrations of plasmid DNA.
The plasmid extraction process is shortened and cost-reduced. The extracted DNA concentration reaches 1.35μg/μL, which can be directly used in the transformation experiment of protoplasts.
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Figure CN119752880A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a high-concentration plasmid extraction method based on a kit. Background Art
[0002] Plasmid extraction technology is the most commonly used biotechnology of molecular biology. There are various rapid plasmid extraction kits on the market now, but the extracted plasmid concentration is low, which is not conducive to experiments with high plasmid concentration purity requirements such as protoplast transformation. The method of traditional plasmid purification is time-consuming, and the reagent kit of circulating plasmid extraction is more expensive on the market, and the extraction cost is higher. Therefore, it is urgent to extract high-concentration plasmid efficiently and quickly. Therefore, the present invention utilizes the operation basis of the small plasmid extraction kit on the market to improve its operating process and invent a method for plasmid extraction with lower cost. Summary of the invention
[0003] The present invention utilizes a traditional plasmid extraction kit, namely, a plasmid rapid small extraction kit (article number P1001-01) of Meiji Biological, and optimizes its operation steps, aiming to increase its plasmid extraction concentration to meet the needs of protoplast transformation experiments.
[0004] To achieve the above-mentioned purpose, the present invention adopts the following technical solutions:
[0005] A method for extracting high-concentration plasmids based on a plasmid rapid mini-extraction kit, characterized in that the method is based on the plasmid rapid mini-extraction kit of Meiji Biology with the item number P1001-01, and the high-concentration plasmid is extracted and purified by optimizing its operation steps, and the specific optimization steps include: increasing the original 5-10mL small volume of bacterial solution to a large volume of 100-200mL bacterial solution; replacing the 2mL centrifuge tube in steps 1-3 with a 50mL centrifuge tube, and correspondingly increasing the dosage of Buffer P1, Buffer P2 and Buffer P3; increasing the number of columns in step 6 to 10 (numbered 1-10), and the operations of steps 7-9 remain unchanged; in step 10, adding 120μL of sterile water to the center of the membrane of the column numbered 1, standing for 1min, eluting DNA at 13000×g for 1min, and then adding the eluate to the column numbered 2 to repeat the above operation until the column numbered 10 is eluted to obtain high-concentration plasmid DNA.
[0006] The method specifically comprises the following steps:
[0007] (1) Collect 200 mL of bacterial cells containing plasmid using a 50 mL centrifuge tube. Centrifuge at 13,000 × g for 1 min, discard the culture medium, and aspirate the residual liquid.
[0008] (2) Add 5 mL of Buffer P1 / RNase A mixture and vortex or pipette to fully resuspend the cells;
[0009] (3) Add 5 mL of Buffer P2 to the resuspension and mix by inverting 8 to 10 times or until the cells are completely lysed;
[0010] (4) Add 7 mL of Buffer NP3 and immediately invert 8 to 10 times to completely neutralize the solution;
[0011] (5) Centrifuge at 13,000 × g for 2 min and collect the supernatant;
[0012] (6) Place 10 HiPure DNA Mini Column IVs in collection tubes, numbered 1-10; load the supernatant into the columns numbered 1-10 in sequence, and centrifuge at 13,000 × g for 60 seconds until all the supernatant has passed through the columns;
[0013] (7) Discard the filtrate, put the column back into the collection tube, add 500 μL Buffer PW1 to the column, and centrifuge at 13,000 × g for 60 seconds;
[0014] (8) Discard the filtrate, put the column back into the collection tube, add 500 μL Buffer PW2 to the column, and centrifuge at 13,000 × g for 60 seconds;
[0015] (9) Repeat step (8) and centrifuge at 13,000 × g for 2 min;
[0016] (10) Place the column in a 1.5 mL centrifuge tube, add 120 μL of sterile water to the center of the membrane of column No. 1, let stand for 1 min, and centrifuge at 13,000 × g for 1 min to elute the DNA. Then add the eluate to column No. 2 and repeat the above steps until column No. 10 is eluted to obtain high-concentration plasmid DNA.
[0017] Preferably, in step (1), the plasmid is pCAMBIA1300 plasmid.
[0018] Preferably, in step (1), the bacterial cells containing the plasmid are obtained by culturing for 16-18 hours in a shaking incubator at 37°C and 220-250 rpm.
[0019] On the other hand, the present invention also provides the use of the high-concentration plasmid extracted according to the above method, and the high-concentration plasmid can be directly used in the transformation experiment of protoplasts.
[0020] Preferably, the concentration of the high-concentration plasmid is 1.35 μg / μL.
[0021] The present invention has the following beneficial effects: the present invention further optimizes the experimental steps on the basis of the original kit, adopts a multiple cumulative washing method to extract and purify high-concentration plasmid DNA, shortens the purification time and purification cost, and finally the concentration of the extracted DNA can reach 1.35 μg / μL, and can be directly used in protoplast transformation experiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 This is the difference in plasmid concentration before and after the kit operation steps are optimized.
[0023] Figure 2 The effect of transforming and introducing the purified plasmid pCAMBIA1300 into rice protoplasts. DETAILED DESCRIPTION
[0024] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.
[0025] Example 1
[0026] A method for extracting high-concentration plasmids based on a plasmid rapid extraction kit is based on the plasmid rapid extraction kit of Meiji Biotechnology with the catalog number P1001-01, and its operation steps are optimized to extract and purify high-concentration plasmids, specifically comprising the following steps:
[0027] (1) Collect 200 mL of bacterial cells containing pCAMBIA1300 plasmid using a 50 mL centrifuge tube. Centrifuge at 13,000 × g for 1 min, discard the culture medium, and aspirate the residual liquid.
[0028] (2) Add 5 mL of Buffer P1 / RNase A mixture and vortex or pipette to fully resuspend the cells;
[0029] (3) Add 5 mL of Buffer P2 to the resuspension and mix by inverting 8 to 10 times or until the cells are completely lysed;
[0030] (4) Add 7 mL of Buffer NP3 and immediately invert 8 to 10 times to completely neutralize the solution;
[0031] (5) Centrifuge at 13,000 × g for 2 min and collect the supernatant;
[0032] (6) Place 10 HiPure DNA Mini Column IVs in collection tubes, numbered 1-10; load the supernatant into the columns numbered 1-10 in sequence, and centrifuge at 13,000 × g for 60 seconds until all the supernatant has passed through the columns;
[0033] (7) Discard the filtrate, put the column back into the collection tube, add 500 μL Buffer PW1 to the column, and centrifuge at 13,000 × g for 60 seconds;
[0034] (8) Discard the filtrate, put the column back into the collection tube, add 500 μL Buffer PW2 to the column, and centrifuge at 13,000 × g for 60 seconds;
[0035] (9) Repeat step (8) and centrifuge at 13,000 × g for 2 min;
[0036] (10) Place the column in a 1.5 mL centrifuge tube, add 120 μL of sterile water to the center of the membrane of column No. 1, let stand for 1 min, centrifuge at 13,000 × g for 1 min to elute the DNA, then add the eluate to column No. 2 and repeat the above steps until column No. 10 is eluted to obtain high-concentration plasmid DNA. Measure the DNA concentration and store at -20°C for later use.
[0037] Figure 1 Comparison of plasmid concentrations before and after optimization. Figure 1 It can be seen that the concentration of the extracted plasmid after optimization reached 1.35μg / μL, which was significantly higher than the plasmid concentration before optimization.
[0038] The steps before optimization of the present invention are performed based on the operating instructions of the plasmid rapid mini-extraction kit of Meiji Biotechnology.
[0039] Example 2
[0040] Protoplast transformation experiment:
[0041] Take 10 μg of the pCAMBIA1300 plasmid extracted above and add it to about 140 μL of rice protoplasts, then immediately add 150 μL of PEG4000 and mix well. After standing at room temperature for 15 min-20 min, add 600 μL of W5 solution (composition: 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES (pH 5.7), solvent is water) and mix well by inversion; centrifuge horizontally, remove the supernatant, add 600 μL of WI solution (composition: 0.5 M mannitol, 20 mM KCl, 4 mM MES (pH 5.7), solvent is water), culture in the dark at 25°C for 24-48 h, collect the cells by centrifugation, and observe under a microscope.
[0042] Figure 2 It was shown that significant green fluorescence signals could be observed in the cytoplasm and nucleus of the protoplasts, indicating that the plasmid had good purity and could be used for protoplast transformation experiments.
[0043] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.
Claims
1. A method for extracting high-concentration plasmids based on a plasmid rapid small extraction kit, characterized in that: Based on the plasmid rapid extraction kit of Meiji Biotechnology with the catalog number P1001-01, multiple cumulative washing methods were adopted to extract and purify high-concentration plasmids, which specifically include the following steps: (1) Collect 200 mL of bacterial cells containing plasmids using a 50 mL centrifuge tube. Centrifuge at 13,000 × g for 1 min, discard the culture medium, and aspirate the residual liquid. (2) Add 5 mL of Buffer P1 / RNase A mixture and vortex or pipette to fully resuspend the cells; (3) Add 5 mL of Buffer P2 to the resuspension and mix by inverting 8 to 10 times or until the cells are completely lysed; (4) Add 7 mL of Buffer NP3 and immediately invert 8 to 10 times to completely neutralize the solution; (5) Centrifuge at 13,000 × g for 2 min and collect the supernatant; (6) Place 10 HiPure DNAMini Column IVs in collection tubes, numbered 1-10; load the supernatant into the columns numbered 1-10 in sequence, and centrifuge at 13,000 × g for 60 seconds until all the supernatant has passed through the columns; (7) Discard the filtrate, put the column back into the collection tube, add 500 μL Buffer PW1 to the column, and centrifuge at 13,000 × g for 60 seconds; (8) Discard the filtrate, put the column back into the collection tube, add 500 μL Buffer PW2 to the column, and centrifuge at 13,000 × g for 60 seconds; (9) Repeat step (8) and centrifuge at 13,000 × g for 2 min; (10) Place the column in a 1.5 mL centrifuge tube, add 120 μL of sterile water to the center of the membrane of column No. 1, let stand for 1 min, and centrifuge at 13,000 × g for 1 min to elute the DNA. Then add the eluate to column No. 2 and repeat the above steps until column No. 10 is eluted to obtain high-concentration plasmid DNA.
2. The method for extracting high-concentration plasmids based on a plasmid rapid small extraction kit according to claim 1, characterized in that: In step (1), the plasmid is pCAMBIA1300 plasmid.
3. The use of high-concentration plasmids extracted by the method according to claim 1 or 2, characterized in that: The high-concentration plasmid can be directly used in protoplast transformation experiments.
4. The use according to claim 3, characterized in that: The concentration of the high-concentration plasmid was 1.35 μg / μL.