Rapid and simple plasmid extraction kit and extraction method
Through the specific ratio of lysate and neutralizing solution combined with nucleic acid adsorption filter membrane, the problems of long plasmid DNA extraction cycle and cumbersome steps are solved, and the efficient simplification and high purity of plasmid extraction are achieved, which is suitable for high-throughput plasmid screening.
Patent Information
- Application Number
- CN202510561265.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-08-05
AI Technical Summary
The existing plasmid DNA extraction methods have long cycles and cumbersome steps, and are especially not suitable for high-throughput plasmid extraction, resulting in inefficient experimental results.
A specific ratio of lysate and neutralizing solution is used, including lysate of sodium hydroxide, sodium dodecyl sulfate, sodium lauroyl sarcosine and other components, as well as a neutralizing solution of guanidine hydrochloride, isopropanol, glacial acetic acid, sodium lauroyl sarcosine and sodium acetate, combined with a nucleic acid adsorption glass fiber filter membrane, the column can be put on without centrifugation after neutralization, simplifying the steps and shortening the cycle.
The plasmid extraction cycle is significantly shortened, the efficiency of large-batch plasmid screening is improved, the operation steps are simplified, and the high purity and high yield of the plasmid are maintained.
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Figure BDA0005384644750000101
Abstract
Description
Technical Field
[0001] The present invention relates to the field of plasmid extraction, in particular to a rapid and simple plasmid extraction kit and an extraction method. Background Art
[0002] Plasmid DNA is the most common vector in genetic engineering and the most important gene delivery system in gene therapy research. It can deliver target DNA fragments into cells through recombinant DNA technology for reproduction and expression. Plasmid DNA extraction and purification are fundamental steps in molecular biology, encompassing gene cloning, gene sequence analysis, nucleic acid vaccines, gene therapy, and various gene recombination techniques. The efficiency and quality of plasmid DNA extraction are closely and directly linked to the success of subsequent molecular biology experiments (enzyme digestion, PCR amplification, and sequencing).
[0003] The classic method for extracting and purifying plasmid DNA from prokaryotes involves resuspension of the cells, alkaline lysis, and acid neutralization. After centrifugation to remove chromosomal DNA and proteins, a clarified supernatant containing the plasmid DNA is obtained. This supernatant is then purified by column chromatography to obtain high-quality plasmid DNA. Currently, most commercial products are based on the alkaline lysis method. This technique, invented by Birnboim & Doly in 1979, involves a three-solution pretreatment: suspension of the cells, denaturation and lysis with alkaline and SDS, and plasmid renaturation and neutralization. Renaturation and neutralization precipitate proteins and genomic DNA, while the plasmid remains dissolved due to its rapid renaturation. Prolonged high-speed centrifugation removes genomic DNA and proteins, yielding a crude supernatant containing the plasmid, which is then purified by column chromatography. The extraction process typically takes 20 to 60 minutes, resulting in a relatively long experimental cycle and cumbersome operation, significantly impacting the researcher's experimental progress. Eppendorf, a German company, invented a one-step plasmid extraction method based on bacterial lysis with lysozyme. The cells are lysed using a lysis buffer containing lysozyme and detergent. After lysis, the cells can be directly bound to a filter membrane. Supercoiled plasmid DNA is then washed and eluted by rapid centrifugation. While this extraction method is convenient, it is costly and limited in versatility, making it unpopular with many customers.
[0004] In high-throughput drug screening and library construction, large-scale plasmid extraction is often required. To speed up the experimental process, researchers need to culture bacteria in 96-well or 384-well plates and perform high-throughput plasmid extraction. Plasmid extraction methods based on one-step methods are not suitable for high-throughput plasmid extraction because they require centrifugal force and precise operation time. Plasmid purification technology based on alkaline lysis produces a large amount of precipitate during neutralization and renaturation, and due to the low centrifugal force of 96 wells, it takes longer to centrifuge to obtain a clear supernatant. After centrifugation, a large amount of loose sediment is formed, resulting in unstable product purity.
[0005] It can be seen that the current methods and products for extracting plasmids have problems such as long plasmid DNA extraction experimental cycle and many operation steps. Therefore, there is still room for improvement. Summary of the Invention
[0006] In order to shorten the cycle of plasmid DNA extraction experiments and simplify the steps, the present application provides a quick and simple plasmid extraction kit and extraction method.
[0007] In the first aspect, the present application provides a rapid and simple plasmid extraction kit, which adopts the following technical solutions: A quick and simple plasmid extraction kit, comprising a resuspension solution, a lysis solution, a neutralization solution, a rinsing solution, and an elution solution; The resuspension solution includes Tris buffer, disodium EDTA, RNase A, and water; The lysis solution includes sodium hydroxide, sodium lauryl sulfate, sodium lauroyl sarcosinate, and water; The neutralizing solution includes guanidine hydrochloride, isopropyl alcohol, glacial acetic acid, sodium lauroyl sarcosinate, sodium acetate, and water; The rinse solution includes sodium chloride, Tris buffer, ethanol, and water; The eluent includes Tris buffer and water.
[0008] By adopting the above technical solution, a lysis step is performed using a lysis solution prepared from sodium hydroxide, sodium lauryl sulfate, sodium lauroyl sarcosinate, and water, and then a neutralization step is performed using a neutralization solution prepared from guanidine hydrochloride, isopropyl alcohol, glacial acetic acid, sodium lauroyl sarcosinate, sodium acetate, and water. This avoids the formation of a large amount of precipitate during the neutralization and renaturation process, enables column loading after neutralization without the need for centrifugation, significantly shortens the plasmid extraction cycle, and improves the efficiency of large-scale plasmid screening.
[0009] Preferably, in the lysis solution, the content of sodium hydroxide is 0.18-0.22 mol / L, the mass content of sodium lauryl sulfate is 0.45-0.55%, and the mass content of sodium lauroyl sarcosinate is 0.45-0.55%.
[0010] By adopting the above technical solution and specifically selecting the content of each component in the lysate, the effect of reducing precipitation is better, and the effects of shortening the experimental cycle and simplifying the steps are better achieved.
[0011] Preferably, in the neutralization solution, the content of guanidine hydrochloride is 2-3 mol / L, the mass content of isopropyl alcohol is 40-50%, the mass content of glacial acetic acid is 1-4%, the mass content of sodium lauroyl sarcosinate is 0.8-1.2%, and the content of sodium acetate is 0.2-0.3 mol / L.
[0012] By adopting the above technical solution and specifically selecting the content of each component in the neutralization solution, the effect of reducing precipitation can be better achieved, which is more conducive to shortening the experimental cycle and simplifying the experimental steps.
[0013] Preferably, in the resuspension, the content of Tris buffer is 10-20 mmol / L, the content of disodium EDTA is 5-10 mmol / L, and the content of RNase A is 95-105 μg / mL.
[0014] By adopting the above technical solution and specifically selecting the content of each component in the resuspension, the effect of plasmid extraction is improved.
[0015] Preferably, the content of sodium chloride in the rinsing solution is 18-22 mmol / L, the content of Tris buffer is 4-6 mmol / L, and the mass content of ethanol is 78-82%.
[0016] By adopting the above technical solution and specifically selecting the content of each component in the rinsing liquid, the plasmid extraction effect is improved and the purity is higher.
[0017] Preferably, the content of Tris buffer in the eluent is 8-12 mmol / L.
[0018] By adopting the above technical solution and specifically selecting the content of each component in the eluent, the elution effect is better, the yield is higher, and the economic value is higher.
[0019] In a second aspect, the present application provides a plasmid extraction method, which adopts the following technical solution: A plasmid extraction method using the above-mentioned rapid and simple plasmid extraction kit comprises the following steps: Step 1), centrifuge the bacterial solution, discard the supernatant, and retain the bacterial cells; Step 2), adding a resuspension solution to the bacterial cells, vortexing and resuspending the bacterial cells to obtain a resuspended bacterial cell solution; Step 3), adding lysis solution to the resuspended bacterial liquid, inverting and mixing to lyse the bacterial cells, and obtaining a lysed bacterial liquid; Step 4), adding a neutralizing solution to the lysed bacterial cell solution, inverting and mixing to neutralize, to obtain a neutralized bacterial cell solution; Step 5), transfer the neutralized bacterial liquid to a purification column and centrifuge; Step 6), adding a rinse solution to the purification column for rinsing and drying; Step 7), add elution buffer to the purification column to elute the plasmid DNA.
[0020] By adopting the above technical solution, the column can be loaded without centrifugation after neutralization, which reduces the steps of centrifuging the neutralization solution before loading it into the column in the traditional process, simplifies the experimental steps, shortens the plasmid extraction cycle, and has high economic value.
[0021] Preferably, the purification column comprises a glass fiber filter membrane with nucleic acid adsorption properties.
[0022] By adopting the above technical solution and specifically selecting a glass fiber filter membrane with nucleic acid adsorption in the purification column, the purification effect is better, the plasmid purity is higher, and the quality is better.
[0023] In summary, this application has the following beneficial effects: 1. Since the present application adopts a lysis solution composed of sodium hydroxide, sodium lauryl sulfate, sodium lauroyl sarcosinate, and water to perform the lysis step, and then uses a neutralization solution composed of guanidine hydrochloride, isopropyl alcohol, glacial acetic acid, sodium lauroyl sarcosinate, sodium acetate, and water to perform the neutralization step, the formation of a large amount of precipitate during the neutralization and renaturation process is avoided, and the column can be loaded without centrifugation after neutralization, which significantly compresses the plasmid extraction cycle and improves the efficiency of large-scale plasmid screening.
[0024] 2. In this application, it is preferred to specifically select the content of each component in the lysate to achieve a better effect of reducing precipitation, thereby better achieving the effect of shortening the experimental cycle and simplifying the steps.
[0025] 3. In this application, it is preferred to specifically select the content of each component in the neutralization solution to better achieve the effect of reducing precipitation, which is more conducive to shortening the experimental cycle and simplifying the experimental steps. DETAILED DESCRIPTION
[0026] The present application is further described in detail below with reference to the embodiments.
[0027] Example 1 A quick and easy plasmid extraction kit comprises a resuspension solution, a lysis solution, a neutralization solution, a rinsing solution and an elution solution.
[0028] The resuspension solution is prepared by mixing Tris buffer, disodium EDTA, RNase A and water.
[0029] The lysis solution is prepared by mixing sodium hydroxide, sodium lauryl sulfate, sodium lauroyl sarcosinate and water.
[0030] The neutralizing solution is prepared by mixing guanidine hydrochloride, isopropyl alcohol, glacial acetic acid, sodium lauroyl sarcosinate, sodium acetate and water.
[0031] The rinse solution is prepared by mixing sodium chloride, Tris buffer, ethanol and water.
[0032] The eluent consisted of Tris buffer and water.
[0033] In the resuspension, the content of Tris buffer is 10 mmol / L, the content of disodium EDTA is 5 mmol / L, the content of RNase A is 95 μg / mL, and water is used as the solvent.
[0034] In the lysis solution, the content of sodium hydroxide is 0.18 mol / L, the mass content of sodium lauryl sulfate is 0.45%, the mass content of sodium lauroyl sarcosinate is 0.45%, and water is used as the solvent.
[0035] In the neutralization solution, the content of guanidine hydrochloride is 2 mol / L, the mass content of isopropyl alcohol is 40%, the mass content of glacial acetic acid is 1%, the mass content of sodium lauroyl sarcosinate is 0.8%, the content of sodium acetate is 0.2 mol / L, water is used as the solvent, and the pH is 4.0.
[0036] The rinsing solution contains 18 mmol / L of sodium chloride, 4 mmol / L of Tris buffer, 78% by mass of ethanol, water as solvent, and a pH of 7.4.
[0037] In the eluent, the content of Tris buffer was 8 mmol / L, water was used as solvent, and the pH was 8.5.
[0038] Tris buffer was purchased from Sangon Biotech (Shanghai) Co., Ltd.
[0039] Disodium EDTA was purchased from Sangon Biotech (Shanghai) Co., Ltd.
[0040] RNase A was purchased from Sangon Biotech (Shanghai) Co., Ltd.
[0041] Sodium hydroxide was purchased from Guangzhou Chemical Reagent Factory.
[0042] Sodium lauryl sulfate was purchased from Sangon Biotech (Shanghai) Co., Ltd.
[0043] Sodium lauroyl sarcosinate was purchased from Sangon Biotech (Shanghai) Co., Ltd.
[0044] Guanidine hydrochloride was purchased from Sangon Biotech (Shanghai) Co., Ltd.
[0045] Isopropyl alcohol was purchased from Guangzhou Chemical Reagent Factory.
[0046] Glacial acetic acid was purchased from Guangzhou Chemical Reagent Factory.
[0047] Sodium acetate was purchased from Guangzhou Chemical Reagent Factory.
[0048] Sodium chloride was purchased from Guangzhou Chemical Reagent Factory.
[0049] Ethanol was purchased from Guangzhou Chemical Reagent Factory, anhydrous ethanol.
[0050] Example 2 A quick and easy plasmid extraction kit, compared with Example 1, differs only in that: In the resuspension, the content of Tris buffer is 15 mmol / L, the content of disodium EDTA is 7.5 mmol / L, the content of RNase A is 100 μg / mL, and water is used as the solvent.
[0051] In the lysis solution, the content of sodium hydroxide is 0.2 mol / L, the mass content of sodium lauryl sulfate is 0.5%, the mass content of sodium lauroyl sarcosinate is 0.5%, and water is used as the solvent.
[0052] In the neutralization solution, the content of guanidine hydrochloride is 2.5 mol / L, the mass content of isopropyl alcohol is 45%, the mass content of glacial acetic acid is 2.5%, the mass content of sodium lauroyl sarcosinate is 1%, the content of sodium acetate is 0.25 mol / L, water is used as the solvent, and the pH is 4.0.
[0053] The rinsing solution contains 20 mmol / L of sodium chloride, 5 mmol / L of Tris buffer, 80% by mass of ethanol, water as solvent, and a pH of 7.4.
[0054] In the eluent, the content of Tris buffer was 10 mmol / L, water was used as solvent, and the pH was 8.5.
[0055] Example 3 A quick and easy plasmid extraction kit, compared with Example 1, differs only in that: In the resuspension, the content of Tris buffer is 20 mmol / L, the content of disodium EDTA is 10 mmol / L, the content of RNase A is 105 μg / mL, and water is used as the solvent.
[0056] In the lysis solution, the content of sodium hydroxide is 0.22 mol / L, the mass content of sodium lauryl sulfate is 0.55%, the mass content of sodium lauroyl sarcosinate is 0.55%, and water is used as the solvent.
[0057] In the neutralization solution, the content of guanidine hydrochloride is 3 mol / L, the mass content of isopropyl alcohol is 50%, the mass content of glacial acetic acid is 4%, the mass content of sodium lauroyl sarcosinate is 1.2%, the content of sodium acetate is 0.3 mol / L, water is used as the solvent, and the pH is 4.0.
[0058] The rinsing solution contains 22 mmol / L of sodium chloride, 6 mmol / L of Tris buffer, 82% by mass of ethanol, water as solvent, and a pH of 7.4.
[0059] In the eluent, the content of Tris buffer was 12 mmol / L, water was used as solvent, and the pH was 8.5.
[0060] Comparative Example 1 A quick and easy plasmid extraction kit, compared with Example 2, differs only in that: The lysis solution is prepared by mixing sodium hydroxide, sodium lauryl sulfate and water.
[0061] In the lysis solution, the content of sodium hydroxide is 0.2 mol / L, the mass content of sodium lauryl sulfate is 1%, and water is used as the solvent.
[0062] Comparative Example 2 A quick and easy plasmid extraction kit, compared with Example 2, differs only in that: The lysis solution is prepared by mixing sodium hydroxide, sodium lauryl sulfate, Triton X100 and water.
[0063] In the lysis solution, the content of sodium hydroxide is 0.2 mol / L, the mass content of sodium lauryl sulfate is 0.5%, the mass content of Triton X100 is 0.5%, and water is used as the solvent.
[0064] Comparative Example 3 A quick and easy plasmid extraction kit, compared with Example 2, differs only in that: The lysis solution is prepared by mixing sodium hydroxide, sodium lauryl sulfate, Tween 20 and water.
[0065] In the lysis solution, the content of sodium hydroxide is 0.2 mol / L, the mass content of sodium lauryl sulfate is 0.5%, the mass content of Tween 20 is 0.5%, and water is used as the solvent.
[0066] Comparative Example 4 A quick and easy plasmid extraction kit, compared with Example 2, differs only in that: The lysis solution is prepared by mixing sodium hydroxide, sodium lauryl sulfate, sodium lauryl sarcosine and water.
[0067] In the lysis solution, the content of sodium hydroxide is 0.2 mol / L, the mass content of sodium lauryl sulfate is 0.5%, the mass content of sodium lauryl sarcosinate is 0.5%, and water is used as the solvent.
[0068] Comparative Example 5 A quick and easy plasmid extraction kit, compared with Example 2, differs only in that: The lysis solution is prepared by mixing sodium hydroxide, sodium lauryl sulfate, alkyl alcohol polyoxyethylene (25) ether and water.
[0069] In the lysis solution, the content of sodium hydroxide is 0.2 mol / L, the mass content of sodium lauryl sulfate is 0.5%, the mass content of alkyl alcohol polyoxyethylene (25) ether is 0.5%, and water is the solvent.
[0070] Comparative Example 6 A quick and easy plasmid extraction kit, compared with Example 2, differs only in that: The lysis solution is prepared by mixing sodium hydroxide, sodium lauryl sulfate, sodium lauroyl sarcosinate and water.
[0071] In the lysis solution, the content of sodium hydroxide is 0.2 mol / L, the mass content of sodium lauryl sulfate is 0.5%, the mass content of sodium lauroyl sarcosinate is 0.25%, and water is used as the solvent.
[0072] Comparative Example 7 A quick and easy plasmid extraction kit, compared with Example 2, differs only in that: The lysis solution is prepared by mixing sodium hydroxide, sodium lauryl sulfate, sodium lauroyl sarcosinate and water.
[0073] In the lysis solution, the content of sodium hydroxide is 0.2 mol / L, the mass content of sodium lauryl sulfate is 0.5%, the mass content of sodium lauroyl sarcosinate is 1%, and water is used as the solvent.
[0074] Comparative Example 8 A quick and easy plasmid extraction kit, compared with Example 2, differs only in that: The lysis solution is prepared by mixing sodium hydroxide, sodium lauryl sulfate, sodium lauroyl sarcosinate and water.
[0075] In the lysis solution, the content of sodium hydroxide is 0.2 mol / L, the mass content of sodium lauryl sulfate is 0.5%, the mass content of sodium lauroyl sarcosinate is 2%, and water is used as a solvent.
[0076] Comparative Example 9 A quick and easy plasmid extraction kit, compared with Example 2, differs only in that: The neutralizing solution is prepared by mixing guanidine hydrochloride, isopropyl alcohol, glacial acetic acid, sodium acetate and water.
[0077] In the neutralization solution, the content of guanidine hydrochloride is 2.5 mol / L, the mass content of isopropyl alcohol is 45%, the mass content of glacial acetic acid is 2.5%, the content of sodium acetate is 0.25 mol / L, water is used as the solvent, and the pH is 4.0.
[0078] Comparative Example 10 A quick and easy plasmid extraction kit, compared with Example 2, differs only in that: The neutralizing solution is prepared by mixing guanidine hydrochloride, isopropyl alcohol, glacial acetic acid, Triton X100, sodium acetate and water.
[0079] In the neutralization solution, the content of guanidine hydrochloride is 2.5 mol / L, the mass content of isopropyl alcohol is 45%, the mass content of glacial acetic acid is 2.5%, the mass content of Triton X100 is 1%, the content of sodium acetate is 0.25 mol / L, water is used as the solvent, and the pH is 4.0.
[0080] Comparative Example 11 A quick and easy plasmid extraction kit, compared with Example 2, differs only in that: The neutralizing solution is prepared by mixing guanidine hydrochloride, isopropyl alcohol, glacial acetic acid, Tween 20, sodium acetate and water.
[0081] In the neutralization solution, the content of guanidine hydrochloride is 2.5 mol / L, the mass content of isopropyl alcohol is 45%, the mass content of glacial acetic acid is 2.5%, the mass content of Tween 20 is 1%, the content of sodium acetate is 0.25 mol / L, water is used as the solvent, and the pH is 4.0.
[0082] Comparative Example 12 A quick and easy plasmid extraction kit, compared with Example 2, differs only in that: The neutralizing solution is prepared by mixing guanidine hydrochloride, isopropyl alcohol, glacial acetic acid, sodium lauryl sarcosine, sodium acetate and water.
[0083] In the neutralization solution, the content of guanidine hydrochloride is 2.5 mol / L, the mass content of isopropyl alcohol is 45%, the mass content of glacial acetic acid is 2.5%, the mass content of sodium lauryl sarcosinate is 1%, the content of sodium acetate is 0.25 mol / L, water is used as the solvent, and the pH is 4.0.
[0084] Comparative Example 13 A quick and easy plasmid extraction kit, compared with Example 2, differs only in that: The neutralizing solution is prepared by compounding guanidine hydrochloride, isopropyl alcohol, glacial acetic acid, alkyl alcohol polyoxyethylene (25) ether, sodium acetate and water.
[0085] In the neutralization solution, the content of guanidine hydrochloride is 2.5 mol / L, the mass content of isopropyl alcohol is 45%, the mass content of glacial acetic acid is 2.5%, the mass content of alkyl alcohol polyoxyethylene (25) ether is 1%, the content of sodium acetate is 0.25 mol / L, water is used as the solvent, and the pH is 4.0.
[0086] Comparative Example 14 A quick and easy plasmid extraction kit, compared with Example 2, differs only in that: The neutralizing solution is prepared by mixing guanidine hydrochloride, isopropyl alcohol, glacial acetic acid, sodium lauroyl sarcosinate, sodium acetate and water.
[0087] In the neutralization solution, the content of guanidine hydrochloride is 2.5 mol / L, the mass content of isopropyl alcohol is 45%, the mass content of glacial acetic acid is 2.5%, the mass content of sodium lauroyl sarcosinate is 0.25%, the content of sodium acetate is 0.25 mol / L, water is used as the solvent, and the pH is 4.0.
[0088] Comparative Example 15 A quick and easy plasmid extraction kit, compared with Example 2, differs only in that: The neutralizing solution is prepared by mixing guanidine hydrochloride, isopropyl alcohol, glacial acetic acid, sodium lauroyl sarcosinate, sodium acetate and water.
[0089] In the neutralization solution, the content of guanidine hydrochloride is 2.5 mol / L, the mass content of isopropyl alcohol is 45%, the mass content of glacial acetic acid is 2.5%, the mass content of sodium lauroyl sarcosinate is 0.5%, the content of sodium acetate is 0.25 mol / L, water is used as the solvent, and the pH is 4.0.
[0090] Comparative Example 16 A quick and easy plasmid extraction kit, compared with Example 2, differs only in that: The neutralizing solution is prepared by mixing guanidine hydrochloride, isopropyl alcohol, glacial acetic acid, sodium lauroyl sarcosinate, sodium acetate and water.
[0091] In the neutralization solution, the content of guanidine hydrochloride is 2.5 mol / L, the mass content of isopropyl alcohol is 45%, the mass content of glacial acetic acid is 2.5%, the mass content of sodium lauroyl sarcosinate is 2%, the content of sodium acetate is 0.25 mol / L, water is used as the solvent, and the pH is 4.0.
[0092] The source information of the raw materials used for replacement in each comparative example is as follows: Triton X100 was purchased from Sangon Biotech (Shanghai) Co., Ltd.
[0093] Tween 20 was purchased from Sangon Biotech (Shanghai) Co., Ltd.
[0094] Sodium lauryl sarcosine was purchased from Sangon Biotech (Shanghai) Co., Ltd.
[0095] Alkyl alcohol polyoxyethylene (25) ether was purchased from Aladdin Biochemical Technology Co., Ltd.
[0096] Experimental example A plasmid extraction method using a rapid and simple plasmid extraction kit comprises the following steps: Step 1) Take 5 ml of overnight cultured Escherichia coli, centrifuge at 10,000 x g for 1 minute, discard the supernatant, and retain the bacteria.
[0097] Step 2) Add 200 μl of resuspension solution to the bacteria, vortex and resuspend the bacteria to obtain a resuspended bacterial solution.
[0098] Step 3) Add 200 μl of lysis buffer to the resuspended bacterial solution, invert and mix to lyse the bacteria to obtain a lysed bacterial solution.
[0099] Step 4) Add 400 μl of neutralizing solution to the lysed bacterial solution, invert and mix to neutralize, and obtain a neutralized bacterial solution.
[0100] Step 5) Transfer the neutralized bacterial liquid to a purification column, centrifuge at 13,000 x g for 1 minute, and discard the waste liquid.
[0101] Step 6) Add 800 μl of rinsing solution to the purification column for rinsing, centrifuge at 13,000×g for 1 minute, and discard the waste liquid.
[0102] Step 7), centrifuge the empty column at 13000xg for 1 minute.
[0103] Step 8) Place the column in a clean centrifuge tube and add 1000 μl of elution buffer to the purification column to elute the plasmid DNA.
[0104] The purification column was purchased from Guangzhou Kangyang Biotechnology Co., Ltd., specification model: ZS-P1001-02.
[0105] Experiment 1 The rapid and simple plasmid extraction kits of the embodiments and comparative examples were used to extract plasmids using the methods of the experimental examples.
[0106] The extracted plasmid was tested for nucleic acid concentration, nucleic acid yield, and nucleic acid purity.
[0107] Detection method: Step 1) Open the Nanodrop software and select the "Nucleic Acid" mode.
[0108] Step 2) Wipe the upper and lower detection bases with a dust-free paper dipped in ultrapure water to ensure there is no residue.
[0109] In step 3), add 1 μL of ultrapure water (or the buffer used, such as TE) to the base and close the base. Click "Blank" to calibrate. When finished, wipe the base dry.
[0110] In step 4), add 1 μL of DNA sample to the assay base and close the base. Click "Measure" to begin the assay. The software will automatically display the concentration (ng / μL) and purity (A260 / A280, A260 / A230).
[0111] Step 5), record the nucleic acid concentration, A260 / A280, and A260 / A230.
[0112] Yield calculation: Nucleic acid yield (μg) = nucleic acid concentration (ng / μL) × elution volume (μL) ÷ 1000 Purity index: The A260 / A280 of pure DNA is 1.8-2.0 (lower than 1.8 indicates protein contamination, higher than 2.0 may contain RNA or phenols).
[0113] The A260 / A230 of pure DNA is 2.0-2.2 (lower than 1.8 indicates residual salt, guanidine salt or organic solvent).
[0114] Pure DNA has a single peak at 260nm. The appearance of other peaks (such as an increase at 280nm) indicates contamination.
[0115] The experimental data are shown in Table 1.
[0116] According to the comparison of the data of each embodiment and the comparative example in Table 1, since each embodiment uses a lysis solution composed of sodium hydroxide, sodium lauryl sulfate, sodium lauroyl sarcosinate, and water, and a neutralization solution composed of guanidine hydrochloride, isopropyl alcohol, glacial acetic acid, sodium lauroyl sarcosinate, sodium acetate, and water, in the process of directly loading the column after omitting the centrifugation step after neutralization, the yield and purity of the extracted plasmid are high, and there is almost no contamination by salts, organic solvents (such as phenol, ethanol), or carbohydrates.
[0117] However, since the special formulation of the lysis solution or neutralization solution was destroyed in each comparative example, the yield and purity of each embodiment were both high, and there was almost no contamination by salts, organic solvents (such as phenol, ethanol) or carbohydrates.
[0118] However, when the sodium lauroyl sarcosinate content in the lysis buffer or neutralization solution is too low, the purification column will be clogged, resulting in a decrease in yield. When the sodium lauroyl sarcosinate content is too high, the A260 / A230 ratio will be too high, and the purity of the nucleic acid will decrease.
[0119] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.
Claims
1. A rapid and simple plasmid extraction kit, characterized by: Including resuspension solution, lysis solution, neutralization solution, rinsing solution, and elution solution; The resuspension solution includes Tris buffer, disodium EDTA, RNase A, and water; The lysis solution includes sodium hydroxide, sodium lauryl sulfate, sodium lauroyl sarcosinate, and water; The neutralizing solution includes guanidine hydrochloride, isopropyl alcohol, glacial acetic acid, sodium lauroyl sarcosinate, sodium acetate, and water; The rinse solution includes sodium chloride, Tris buffer, ethanol, and water; The eluent includes Tris buffer and water.
2. A rapid and simple plasmid extraction kit according to claim 1, characterized in that: In the lysate, the content of sodium hydroxide is 0.18-0.22 mol / L, the mass content of sodium lauryl sulfate is 0.45-0.55%, and the mass content of sodium lauroyl sarcosinate is 0.45-0.55%.
3. A rapid and simple plasmid extraction kit according to claim 2, characterized in that: In the neutralization solution, the content of guanidine hydrochloride is 2-3 mol / L, the mass content of isopropyl alcohol is 40-50%, the mass content of glacial acetic acid is 1-4%, the mass content of sodium lauroyl sarcosinate is 0.8-1.2%, and the content of sodium acetate is 0.2-0.3 mol / L.
4. A rapid and simple plasmid extraction kit according to claim 3, characterized in that: In the resuspension, the content of Tris buffer is 10-20 mmol / L, the content of disodium EDTA is 5-10 mmol / L, and the content of RNase A is 95-105 μg / mL.
5. A rapid and simple plasmid extraction kit according to claim 4, characterized in that: The rinsing solution contains 18-22 mmol / L of sodium chloride, 4-6 mmol / L of Tris buffer, and 78-82% by weight of ethanol.
6. A rapid and simple plasmid extraction kit according to claim 5, characterized in that: In the eluent, the content of Tris buffer is 8-12 mmol / L.
7. A method for extracting a plasmid using the rapid and simple plasmid extraction kit according to any one of claims 1 to 6, characterized in that: The following steps are involved: Step 1) centrifuge the bacterial solution, discard the supernatant, and retain the bacterial cells; Step 2), adding a resuspension solution to the bacteria, vortexing and resuspending the bacteria to obtain a resuspended bacteria solution; Step 3) adding lysis solution to the resuspended bacterial solution, inverting and mixing to lyse the bacteria to obtain a lysed bacterial solution; Step 4), adding neutralizing solution to the lysed bacterial cell solution, inverting and mixing to neutralize, to obtain a neutralized bacterial cell solution; Step 5), transfer the neutralized bacterial liquid to a purification column and centrifuge; Step 6), adding a rinse solution to the purification column for rinsing and drying; In step 7, add elution buffer to the purification column to elute the plasmid DNA.
8. A plasmid extraction method according to claim 7, characterized in that: The purification column comprises a glass fiber filter membrane with nucleic acid adsorption capability.