A plasmid extractor and extraction method

Abstract

The invention discloses a plasmid extractor and an extraction method. The plasmid extractor comprises a transparent tube with external threads at both ends, a first cover body with internal threads on the inner surface of the side wall, and a second cover body with internal threads on the inner surface of the side wall; a first connecting tube is provided on the top wall penetrating the first cover body; a second connecting tube is provided on the bottom wall penetrating the second cover body; a slow qualitative filter paper and a medical gauze sheet are movably connected to the inner surface of the top wall of the first cover body in sequence; a support and a microporous filter membrane are movably connected to the inner surface of the bottom wall of the second cover body in sequence; the first cover body is threadedly connected to the end of the transparent tube; and the second cover body is threadedly connected to the other end of the transparent tube. The plasmid extractor of the invention, combined with a nucleic acid extraction vacuum purification filtration system, can realize rapid plasmid extraction, complete the entire process from collecting bacteria to extracting plasmids within at least 15 minutes, is simple and convenient to operate, greatly saves time, reduces labor intensity, and has good quality of extracted plasmids.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a serial plasmid rapid device and an extraction method. Background Art

[0002] Plasmid extraction is a basic operation in biomedical research. The entire process of traditional plasmid extraction includes shaking bacteria, collecting bacteria, extracting plasmids, etc. At present, most laboratories use adsorption column plasmid extraction kits to assist the plasmid extraction process. The operator's labor is mainly reflected in the process of collecting bacteria and plasmid extraction. The process requires multiple centrifugation (8-9 times), the process is repetitive and cumbersome. In order to reduce the labor intensity of the operator, a simple and easy method for rapid plasmid extraction is urgently needed. Summary of the invention

[0003] The technical solution of the present invention is summarized as follows:

[0004] A plasmid extractor comprises a transparent tube 1 with external threads at both ends, a first cover body 2 with internal threads on the inner surface of the side wall, and a second cover body 3 with internal threads on the inner surface of the side wall; a first connecting tube 4 is provided through the top wall of the first cover body 2; a second connecting tube 9 is provided through the bottom wall of the second cover body 3; a slow qualitative filter paper 5 and a medical gauze sheet 6 are movably connected in sequence to the inner surface of the top wall of the first cover body 2; a support 8 and a microporous filter membrane 7 with a pore size of 0.2-0.45 μm are movably connected in sequence to the inner surface of the bottom wall of the second cover body 3; the first cover body 2 is threadedly connected to the end of the transparent tube 1; and the second cover body 3 is threadedly connected to the other end of the transparent tube 1.

[0005] The support 8 is preferably medical gauze, non-woven fabric or filter paper.

[0006] A plasmid extraction method comprises the following steps:

[0007] 1) Shake the bacteria overnight;

[0008] 2) Using the plasmid extractor of claim 1, the plasmid extractor comprises a transparent tube 1 with external threads at both ends, a first cover body 2 with internal threads on the inner surface of the side wall, and a second cover body 3 with internal threads on the inner surface of the side wall; a first connecting tube 4 is provided through the top wall of the first cover body 2; a second connecting tube 9 is provided through the bottom wall of the second cover body 3; a slow qualitative filter paper 5 and a medical gauze sheet 6 are movably connected to the inner surface of the top wall of the first cover body 2 in sequence; a support 8 and a microporous filter membrane 7 with a pore size of 0.2-0.45 μm are movably connected to the inner surface of the bottom wall of the second cover body 3 in sequence; the first cover body 2 is threadedly connected to the end of the transparent tube 1; the second cover body 3 is threadedly connected to the other end of the transparent tube 1;

[0009] 3) Unscrew the first cover 2, add the bacterial solution obtained in step 1) into the transparent tube; insert the second connecting tube 9 into the adsorption column jack of the nucleic acid extraction vacuum purification filtration system, filter, and leave the bacteria on the microporous filter membrane 7, continue to drain, and remove the plasmid extractor;

[0010] 4) Using a plasmid extraction kit, add solution P1 to a transparent tube, and use a pipette to pump and resuspend the bacteria on the microporous filter membrane 7; add solution P2, mix well, and lyse the bacteria; add solution P3, mix well, and a white flocculent precipitate is generated; insert the adsorption column into the adsorption column jack of the nucleic acid extraction vacuum purification filtration system, add 0.5 mL of balancing solution BL, equilibrate the adsorption column, and drain;

[0011] 5) Tighten the first cover 2 and the transparent tube, turn over the transparent tube so that the first cover 2 is at the bottom, unscrew the second cover 3, insert the first connecting tube 4 connected to the first cover into the adsorption column, and filter; the plasmid is retained on the adsorption membrane of the adsorption column;

[0012] 6) Remove the plasmid extractor and rinse the adsorption column twice with rinsing solution PW to remove impurities on the adsorption membrane; put the adsorption column into the collection tube and then into the centrifuge, centrifuge at 12000rpm for 2min; add 50μL elution buffer EB into the adsorption column, centrifuge at 12000rpm for 2min to elute the plasmid.

[0013] The support 8 is preferably medical gauze, non-woven fabric or filter paper.

[0014] Advantages of the present invention:

[0015] The plasmid extractor of the present invention, combined with the nucleic acid extraction vacuum purification filtration system, can realize rapid plasmid extraction and complete the entire process from bacteria collection to plasmid extraction within 15 minutes. The operation is simple and convenient, which greatly saves the workers' time and reduces their labor intensity, and the extracted plasmid has good quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the dispersion of the plasmid extractor of the present invention.

[0017] Figure 2 The electrophoresis diagram of the plasmid extracted by the method of the present invention. DETAILED DESCRIPTION

[0018] Microporous membrane: Zhejiang Tailin Biotechnology Co., Ltd., Shanghai Xinya Purification Device Factory;

[0019] Haimen Chunbo Biological Experiment Equipment Factory, Haimen Chunbo Biological Experiment Equipment Factory;

[0020] Nucleic acid extraction vacuum purification filtration system: American Shengster Company, Shanghai Lingde Instrument Co., Ltd.;

[0021] Slow qualitative filter paper: Shanghai Jinpan Biotechnology Co., Ltd., Hangzhou Special Paper Co., Ltd.;

[0022] The plasmid extraction kit includes: balance solution BL, solution P1, solution P2, solution P3, rinse solution PW, elution buffer EB, adsorption column CP4, and collection tube (2 mL).

[0023] The present invention takes DP106-Plasmid Mini-Extraction Medium-Quantity Kit (centrifugal column type): Tiangen Biochemical Technology (Beijing) Co., Ltd. as an example.

[0024] The disclosure of the manufacturing companies of the above-mentioned articles is to enable ordinary technicians in the field to better implement the present invention, but does not impose any limitation on the present invention.

[0025] The present invention will be further described below by way of the accompanying drawings and examples.

[0026] A plasmid extractor, see Figure 1 , comprising a transparent tube 1 with external threads at both ends, a first cover body 2 with internal threads on the inner surface of the side wall, and a second cover body 3 with internal threads on the inner surface of the side wall; a first connecting tube 4 is arranged through the top wall of the first cover body 2; a second connecting tube 9 is arranged through the bottom wall of the second cover body 3; a slow qualitative filter paper 5 and a medical gauze sheet 6 are movably connected to the inner surface of the top wall of the first cover body 2 in sequence; a support 8 and a microporous filter membrane 7 with a pore size of 0.2-0.45 μm are movably connected to the inner surface of the bottom wall of the second cover body 3 in sequence; the first cover body 2 is threadedly connected to the end of the transparent tube 1; the second cover body 3 is threadedly connected to the other end of the transparent tube 1.

[0027] Example 1

[0028] A plasmid extraction method comprises the following steps:

[0029] 1) Overnight shaking of bacteria (Escherichia coli DH5α, commercially available);

[0030] 2) A plasmid extractor is used, which includes a transparent tube 1 with external threads at both ends, a first cover body 2 with internal threads on the inner surface of the side wall, and a second cover body 3 with internal threads on the inner surface of the side wall; a first connecting tube 4 is provided through the top wall of the first cover body 2; a second connecting tube 9 is provided through the bottom wall of the second cover body 3; a slow qualitative filter paper 5 and a medical gauze sheet 6 are movably connected to the inner surface of the top wall of the first cover body 2 in sequence; a support 8 (medical gauze, non-woven fabric or filter paper can also be selected) and a microporous filter membrane 7 with a pore size of 0.45 μm (a microporous filter membrane with any pore size between 0.2-0.45 μm, such as 0.2, 0.2, 0.2, 0.42 μm) are movably connected to the inner surface of the bottom wall of the second cover body 3 in sequence; the first cover body 2 is threadedly connected to the end of the transparent tube 1; the second cover body 3 is threadedly connected to the other end of the transparent tube 1;

[0031] 3) Unscrew the first cover 2, add 5 mL of the bacterial solution obtained in step 1) into the transparent tube; insert the second connecting tube 9 connected to the second cover into the adsorption column jack of the nucleic acid extraction vacuum purification filtration system, filter, and leave the bacteria on the microporous filter membrane 7, continue to drain, and remove the plasmid extractor;

[0032] 4) Using a plasmid extraction kit, add 1 mL of solution P1 to a transparent tube, and use a 1 mL pipette to pump and resuspend the bacteria on the microporous filter membrane 7; add 1 mL of solution P2, mix well, and lyse the bacteria; add 1.5 mL of solution P3, mix well, and a white flocculent precipitate is generated; insert the adsorption column into the adsorption column jack of the nucleic acid extraction vacuum purification filtration system, add 0.5 mL of balancing solution BL, balance the adsorption column, and drain;

[0033] 5) Tighten the first cover 2 and the transparent tube, turn over the transparent tube so that the first cover 2 is at the bottom, unscrew the second cover 3, insert the first connecting tube 4 connected to the first cover into the balanced adsorption column, and filter; the plasmid is retained on the adsorption membrane of the adsorption column;

[0034] 6) Remove the plasmid extractor and rinse the adsorption column twice with rinsing solution PW, 0.7 mL each time to remove impurities on the adsorption membrane; put the adsorption column into the collection tube and then into the centrifuge, centrifuge at 12000 rpm for 2 min; add 50 μL of elution buffer EB into the adsorption column, centrifuge at 12000 rpm for 2 min to elute the plasmid. See Table 1 and Figure 2 ; (Concentration detection of the extracted plasmid by agarose gel electrophoresis)

[0035] Table 1

[0036] Time (minutes) Centrifugation times Extraction process Extraction plasmid concentration Traditional centrifugation 50 8 Complicated 0.675 μg / μL Method of the present invention 25 2 Simple 0.914 μg / μL

Claims

1. A plasmid extractor, characterized in that The invention comprises a transparent tube (1) with external threads at both ends, a first cover body (2) with internal threads on the inner surface of the side wall, and a second cover body (3) with internal threads on the inner surface of the side wall; a first connecting tube (4) is provided through the top wall of the first cover body (2); a second connecting tube (9) is provided through the bottom wall of the second cover body (3); a slow qualitative filter paper (5) and a medical gauze sheet (6) are movably connected to the inner surface of the top wall of the first cover body (2); a support (8) and a microporous filter membrane (7) with a pore size of 0.2-0.45 μm are movably connected to the inner surface of the bottom wall of the second cover body (3); the first cover body (2) is threadedly connected to the end of the transparent tube (1); the second cover body (3) is threadedly connected to the other end of the transparent tube (1); the support (8) is medical gauze, non-woven fabric or filter paper.

2. A plasmid extraction method, characterized in that The steps include: 1) Shake the bacteria overnight; 2) Using the plasmid extractor of claim 1, the plasmid extractor comprises a transparent tube (1) with external threads at both ends, a first cover body (2) with internal threads on the inner surface of the side wall, and a second cover body (3) with internal threads on the inner surface of the side wall; a first connecting tube (4) is provided through the top wall of the first cover body (2); a second connecting tube (9) is provided through the bottom wall of the second cover body (3); a slow qualitative filter paper (5) and a medical gauze sheet (6) are movably connected to the inner surface of the top wall of the first cover body (2); a support (8) and a microporous filter membrane (7) with a pore size of 0.2-0.45 μm are movably connected to the inner surface of the bottom wall of the second cover body (3); the first cover body (2) is threadedly connected to the end of the transparent tube (1); the second cover body (3) is threadedly connected to the other end of the transparent tube (1); the support (8) is medical gauze, non-woven fabric or filter paper; 3) Unscrew the first cover (2), add the bacterial solution obtained in step 1) into the transparent tube; insert the second connecting tube (9) into the adsorption column jack of the nucleic acid extraction vacuum purification filtration system, filter, and leave the bacteria on the microporous filter membrane (7), continue to drain, and remove the plasmid extractor; 4) Using a plasmid extraction kit, add solution P1 to a transparent tube, and use a pipette to pump and resuspend the bacteria on the microporous filter membrane (7); add solution P2, mix well, and lyse the bacteria; add solution P3, mix well, and a white flocculent precipitate is generated; insert the adsorption column into the adsorption column jack of the nucleic acid extraction vacuum purification filtration system, add 0.5 mL of balancing solution BL, equilibrate the adsorption column, and drain; 5) Tighten the first cover (2) and the transparent tube, turn over the transparent tube so that the first cover (2) is at the bottom, unscrew the second cover (3), insert the first connecting tube (4) connected to the first cover into the adsorption column, and filter; the plasmid is retained on the adsorption membrane of the adsorption column; 6) Remove the plasmid extractor and rinse the adsorption column twice with solution PW to remove impurities on the adsorption membrane; put the adsorption column into the collection tube and then into the centrifuge, centrifuge at 12000rpm for 2min; add 50μL of elution solution EB into the adsorption column, centrifuge at 12000rpm for 2min to elute the plasmid.

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