Molecular cloning device and molecular cloning method

Abstract

The molecular cloning device comprises a pipetting workstation, a peripheral equipment island platform used for storing and transferring consumables and a moving station capable of interacting samples with a central mechanical arm, the peripheral equipment island platform comprises an island platform mechanical arm, and a PCR instrument, a film sealing and tearing device and a centrifugal machine are arranged at the moving station; the plasmid extraction and cell transformation plate coating workstation is used for extracting plasmids and transforming competent cells for plate coating; the bacterial colony picking instrument is used for picking bacterial colonies in the culture dish into the liquid culture medium of the deep hole plate; the constant-temperature and constant-humidity automatic incubator is used for automatically culturing competent cells under a constant-temperature and constant-humidity condition; and the central mechanical arm is used for transferring samples and / or consumables among the mobile station, the plasmid extraction and cell transformation plate coating workstation, the bacterial colony picking instrument and the constant-temperature and constant-humidity automatic incubator. The automation degree can be improved, and pollution introduction is reduced.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a molecular cloning device and a molecular cloning method. Background Technology

[0002] Molecular cloning refers to the process of linking a target gene with a vector DNA to form a recombinant DNA molecule in vitro, then introducing it into a host cell, allowing the target gene to replicate, amplify, and be expressed within the host cell. Molecular cloning technology is one of the core technologies of modern biotechnology, widely used in gene function research, protein expression and production, gene therapy, genetic breeding, and many other fields, providing a powerful tool for the development of life sciences and the application of biotechnology.

[0003] Currently, most experimental procedures in traditional molecular cloning still require manual operation. Even with the assistance of automated workstations such as pipetting workstations, the experimental process cannot be completed efficiently and quickly, resulting in problems such as easy contamination, easy introduction of impurities, and low efficiency in molecular cloning. In addition, some automated equipment exists, such as an automated functional island. This island contains instruments including an automated robotic arm, an automated pipetting workstation, an automated PCR instrument, an automated centrifuge, a consumables stack, a sealing machine, a tearing machine, an ELISA reader, and an automated shaking incubator. The automated pipetting workstation is equipped with flexible eight-channel pipette tips, high-throughput 96-channel pipette tips, a multi-functional gripper robotic arm, a low-temperature control module, a heating control module, a shaking module, plate holders, pipette holders, and several consumable carriers. Although this device can automate operations such as PCR amplification, plasmid construction, transformation, culture, cloning, and plasmid extraction, some of its components are too large, and some procedures still require manual intervention, making it prone to contamination. Summary of the Invention

[0004] Therefore, it is necessary to provide a molecular cloning device that can improve process automation, reduce contamination introduction, and increase efficiency.

[0005] One embodiment of this application provides a molecular cloning apparatus.

[0006] A molecular cloning device, comprising:

[0007] The device includes a pipetting workstation, an island for storing and transferring consumables, and a mobile station for interacting with a central robotic arm. The island includes an island robotic arm for transferring reagents and consumables. The mobile station is equipped with a sealing and tearing device and a centrifuge for centrifugation.

[0008] The PCR instrument is located at the pipetting workstation or the mobile workstation;

[0009] Plasmid extraction and cell transformation plating workstation, used for extracting plasmids and transforming competent cells into plating plates;

[0010] A colony picker is used to pick up colonies from a petri dish and transfer them into the liquid culture medium in a deep-well plate.

[0011] The temperature and humidity automated incubator is used for the automated culture of competent cells under constant temperature and humidity conditions.

[0012] In addition, a central robotic arm is used to transfer samples and / or consumables between the moving workstation, the plasmid extraction and cell transformation plating workstation, the colony picker, and the constant temperature and humidity automated incubator.

[0013] In some embodiments, the pipetting workstation includes a pipetting stage and a component connected to the pipetting stage:

[0014] The first magnetic frame is used to attract magnetic beads;

[0015] The first metal bath is used to preserve reagents at a constant temperature of 4℃~60℃.

[0016] Opening module, used to open the caps of cryopreservation tubes;

[0017] The first pipette is used for pipetting;

[0018] The first rotating plate gripper is used to transfer container consumables or consumable trays between the moving workstations of the pipetting workstation.

[0019] PCR product separation and purification device, used to separate and purify PCR products;

[0020] The first heating and oscillation module is used to heat the orifice plate and rotate the orifice plate to mix it.

[0021] The dispensing needle is connected to a multi-channel rotary valve for reagent dispensing;

[0022] Waste liquid needle, which is connected to a peristaltic pump, is used to remove waste liquid;

[0023] And a vacuum suction cup, used to open the petri dish lid.

[0024] In some embodiments, the pipetting stage is further provided with a first guide component capable of lateral and longitudinal movement in the horizontal direction, and the capping module, the first pipette, the first rotary plate gripper, the sample needle and the waste needle are respectively disposed on the first guide component.

[0025] In some embodiments, the first guide component includes a first guide rail extending laterally in the horizontal direction, a second guide rail slidably connected to the first guide rail, and a first sliding seat slidably connected to the second guide rail. The cap opening module, the first pipette, the first rotating plate gripper, the sample dispensing needle, and the waste liquid needle are respectively disposed on the first sliding seat.

[0026] In some embodiments, the pipetting workstation further includes a passive consumable tray base connected to the pipetting worktable for storing consumables that do not require power.

[0027] In some embodiments, the pipetting workstation further includes a multi-station tilting stage connected to the pipetting worktable for tilting the orifice plate to concentrate the liquid in the orifice plate to one side edge.

[0028] In some embodiments, the multi-station tilting stage is connected to a tilting stage driving component, the multi-station tilting stage is rotatably connected to the pipetting stage, and the multi-station tilting stage can be rotated to the required tilting angle and maintained under the drive of the tilting stage driving component.

[0029] In some embodiments, the pipetting workstation further includes a trash can guide connected to the pipetting worktable for accommodating and storing waste consumables.

[0030] In some embodiments, the agarose gel electrophoresis apparatus has a double-row comb to allow the first pipette to be guided directly from the second row of wells with visual assistance when electrophoresis reaches the second row of wells to remove the target molecular weight band.

[0031] In some embodiments, the pipetting workstation further includes a needle cleaning module for cleaning the needles and / or waste needles.

[0032] In some embodiments, the capping module can achieve independent Z-axis single-channel rotary capping of cryopreservation tubes. The capping module can move independently in the vertical direction and open or close the cap of one capped cryopreservation tube at a time.

[0033] In some embodiments, the cap opening module includes a first cap opening gripper, a second cap opening gripper, a first cap opening drive component, and a second cap opening drive component. The first cap opening drive component and the second cap opening drive component are respectively installed on the pipetting stage. The first cap opening gripper is connected to the first cap opening drive component. The first cap opening drive component is used to drive the first cap opening gripper to move and clamp the test tube. The second cap opening drive component is connected to the second cap opening gripper to drive the second cap opening gripper to move and clamp the test tube cap. The second cap opening drive component drives the second cap opening gripper to move in the opposite direction to open or close the test tube cap.

[0034] In some embodiments, the first pipette includes a first multi-channel variable-pitch independent Z-axis pipette, which can control each pipette tip to move independently in the vertical direction and can adjust the distance between different channels as needed to adapt to multi-well plates of different sizes.

[0035] In some embodiments, the sampling needle includes an independent Z-axis sampling needle that is capable of moving independently in the vertical direction.

[0036] In some embodiments, the waste needle includes an independent Z-axis waste needle, and the sample dispensing needle is capable of moving independently in the vertical direction.

[0037] In some embodiments, the peripheral device island includes:

[0038] A screw-on test tube opener is used to open test tubes.

[0039] Large-capacity light-shielding high and low temperature chamber, used for incubating samples in well plates and temporarily storing low-temperature reagents;

[0040] Consumable tower, used to store consumables including nozzle boxes and orifice plates;

[0041] In addition, there is a transfer station for interacting with the central robotic arm. The island robotic arm can obtain reagent sample containers or trays containing reagent consumable containers from the screw cap test tube opener and the large-capacity light-shielding high and low temperature chamber and transfer them to the transfer station.

[0042] In some embodiments, the peripheral device island is located downstream of the pipetting workstation.

[0043] In some embodiments, the peripheral equipment island also includes an island workbench, and the island robotic arm is slidably connected to the island robotic arm to transfer reagents and consumables between the large-capacity light-shielding high and low temperature chamber and the consumable tower.

[0044] In some embodiments, the mobile workstations include multiple units, and each of the PCR instrument, the sealing and tearing device, and the centrifuge is respectively installed at a different mobile workstation.

[0045] In some embodiments, the plasmid extraction and cell transformation plate-coating workstation includes:

[0046] The second pipette is used for pipetting;

[0047] The second rotating plate gripper is used to grip and move the transfer device containing the tube of competent cells;

[0048] The second heating and oscillation module is used to heat the porous plate and oscillate and mix it.

[0049] The second magnetic frame is used to attract magnetic beads;

[0050] Plasmid extraction equipment, used for automated plasmid extraction;

[0051] Cell transformation mechanism, used to transform competent cells;

[0052] And, a coating module for coating competent cells.

[0053] In some embodiments, the plasmid extraction device includes:

[0054] Filter column, used to filter cell lysate;

[0055] A collection tube, located below the filter column, is used to collect plasmids filtered by the filter column and purify the plasmids using magnetic beads.

[0056] DNA extraction column, used to enrich plasmids from the collection tube onto a silica membrane after positive pressure;

[0057] Waste liquid tank for collecting waste liquid from the filter column;

[0058] And a plasmid collection plate for collecting plasmids purified by magnetic beads from the collection tube, or plasmids from the silica membrane of the DNA extraction column.

[0059] In some embodiments, the cell transformation mechanism includes a second metal bath for applying a cold or hot bath to competent cell tubes held in place on a transfer fixture.

[0060] In some embodiments, the second metal bath includes a cold metal bath mechanism and a hot metal bath mechanism; the cold metal bath mechanism is used to provide a cold bath for the competent cell tube stuck on the transfer fixture, and the hot metal bath mechanism is used to provide a hot bath for the competent cell tube stuck on the transfer fixture.

[0061] In some embodiments, the cold metal bath mechanism has multiple channel stations, enabling it to be matched to the 96-well throughput of the plasmid collection plate.

[0062] In some embodiments, the second pipette includes a second multi-channel variable-pitch independent Z-axis pipette, which can control each pipette tip to move independently in the vertical direction and can adjust the distance between different channels as needed to accommodate multi-well plates of different sizes.

[0063] In some embodiments, the extraction and conversion coating worktable is further provided with a second guide component capable of moving laterally and longitudinally in the horizontal direction, and the second pipette and the second rotating plate gripper are respectively disposed on the second guide component.

[0064] In some embodiments, the second guide component includes a third guide rail extending laterally in the horizontal direction, a fourth guide rail slidably connected to the third guide rail, and a second sliding seat slidably connected to the fourth guide rail, wherein the second pipette and the second rotary plate gripper are respectively disposed on the second sliding seat.

[0065] In some embodiments, the coating module includes a lifting and rotating module coating head, which is mounted on an indexing plate. The indexing plate is rotatable to move the lifting and rotating module coating head to a cleaning station, a high-temperature infrared sterilization station, and a drying station, and to perform cleaning, high-temperature infrared sterilization, and drying of the lifting and rotating module coating head respectively.

[0066] In some embodiments, the plasmid extraction and cell transformation plating workstation further includes a pipette tip holder for storing pipette tips.

[0067] In some embodiments, the plasmid extraction and cell transformation plating workstation also includes a trash can for storing waste consumables and waste filter columns.

[0068] In some embodiments, the colony picker has a petri dish inlet / outlet for interacting with the central robotic arm to pick up the petri dish containing the colonies; and a culture medium inlet / outlet for interacting with the central robotic arm to store the picked colonies in a culture medium container.

[0069] In some embodiments, the colony picker includes:

[0070] A colony picking workbench, which is used to hold several culture dishes;

[0071] A feeding mechanism is provided on the colony picking workbench and is used to continuously output materials.

[0072] The microbial picking mechanism is located above the colony picking workbench. The microbial picking mechanism is used to receive the material output by the feeding mechanism and drive the head end of the material to output a preset length towards the colony picking workbench to form a microbial picking component for picking microbial bacteria.

[0073] A driving mechanism is connected to the picking mechanism. The driving mechanism is used to drive the picking mechanism to move so that the picking mechanism drives the picking element to pick up colonies from one of the culture dishes, and / or so that the picking mechanism drives the picking element to plant colonies in another culture dish.

[0074] And a cutting mechanism for separating the picker from the material.

[0075] In some embodiments, the feeding mechanism includes:

[0076] A conduit, one end of which is connected to the bacteria-picking mechanism;

[0077] An extrusion mechanism is provided on the colony picking workbench and connected to the other end of the conduit. The extrusion mechanism is used to transport the material along the conduit to the colony picking mechanism.

[0078] In some embodiments, the extrusion mechanism includes: a drive wheel rotatably mounted on the colony-picking worktable and a driven wheel spaced apart from the drive wheel, forming a conveying channel for conveying the material between the driven wheel and the drive wheel, and the drive wheel being power-driven to rotate. Driving the drive wheel enables the drive wheel and the driven wheel to cooperate in driving the material forward.

[0079] In some embodiments, the cutting mechanism includes a cutting blade movably disposed on the colony picking worktable, the cutting blade being operable to cut off the material at the end of the conduit.

[0080] One embodiment of this application also provides a molecular cloning method.

[0081] A molecular cloning method, employing the molecular cloning apparatus of any of the above embodiments, includes the following steps:

[0082] The system controls the robotic arm on the island to transfer the multi-well plate from the sub-zero temperature environment of the peripheral equipment island to the centrifuge at the mobile station, and controls the centrifuge to centrifuge; the robotic arm on the island transfers the multi-well plate to the sealing and tearing device at the mobile station, and controls the sealing and tearing device to remove the sealing film from the multi-well plate; the robotic arm on the island transfers the multi-well plate from the sealing and tearing device to the first metal bath at a first preset temperature in the pipetting workstation; the robotic arm on the island transfers the low-temperature reagent from the peripheral equipment island to the first metal bath; the pipetting workstation adds the required low-temperature reagent to the wells of the multi-well plate, and adds other required reagents to prepare the PCR reaction system; the robotic arm on the island feeds the multi-well plate into the PCR instrument at the mobile station, and controls the PCR instrument to amplify; the PCR amplification product is separated and purified by the agarose gel electrophoresis instrument at the pipetting workstation, and the purified fragment is transferred to a new PCR plate;

[0083] The robotic arm on the island is controlled to transfer the PCR plate containing the purified fragment to the first metal bath at a first preset temperature. The robotic arm also controls the transfer of other enzyme digestion system reagents stored below 0°C from the peripheral device island to the first metal bath. A pipetting station is controlled to transfer enzyme digestion system reagents into the PCR plate to prepare the digestion system, which is then reacted in the first metal bath at a second preset temperature for 2-3 hours. The digestion products are recovered. A ligation reaction system containing the digestion products is constructed and mixed in a PCR tube, then reacted overnight at room temperature for 8-12 hours to obtain a plasmid that integrates the target DNA fragment into the vector plasmid. The plasmid is then extracted.

[0084] The central robotic arm is controlled to deliver a new multi-well plate containing plasmids into the plasmid extraction and cell transformation plating workstation for transformation, and to transfer the transformed competent cells to the culture dishes of the plating module; the plating module is controlled to spread the transformed competent cells evenly and to perform cleaning and sterilization procedures.

[0085] The central robotic arm and the island robotic arm are controlled to work together to invert the coated culture dish and transfer it to an automated incubator with constant temperature and humidity for cultivation.

[0086] After the culture is completed, the central robotic arm is controlled to sequentially send the culture dishes into the colony picker for colony selection. The picked colonies are then stored in a deep-well plate containing culture medium.

[0087] In some embodiments, before the robotic arm of the control island transfers the multiwell plate from the sub-zero temperature environment of the peripheral equipment island to the centrifuge, the following preparations are also included: placing the multiwell plate containing the DNA template and sealed with a membrane and low-temperature reagents into a large-capacity light-shielding high and low temperature chamber, placing room-temperature reagents and consumables into the designated position on the passive consumable plate base, and configuring a PCR instrument, a membrane sealing and tearing device and a centrifuge at the moving work station.

[0088] In some embodiments, when controlling the island robotic arm to transfer the perforated plate from the sub-zero temperature environment of the peripheral equipment island to the centrifuge at the moving station, the specific steps include: controlling the island robotic arm to transfer the perforated plate from the sub-zero temperature environment of the large-capacity light-shielding high and low temperature chamber of the peripheral equipment island to the centrifuge at the moving station.

[0089] In some embodiments, the island robotic arm is controlled to pick up a balancing orifice plate from the consumable tower for balancing the centrifuge.

[0090] In some embodiments, when controlling the pipetting workstation to transfer the required cryogenic reagent into the well of the multi-well plate, the following steps are included: controlling the capping module to unscrew the cap of the cryogenic reagent tube, controlling the first pipette to draw the required cryogenic reagent into the well of the multi-well plate, and controlling the capping module to reseal the cap.

[0091] In some embodiments, when adding other required reagents to the wells of the multi-well plate to prepare the PCR reaction system, the following steps are included: controlling the first pipette to add other required reagents to prepare the PCR reaction system.

[0092] In some embodiments, when separating and purifying PCR amplification products using an agarose gel electrophoresis apparatus at a pipetting workstation, and transferring the purified fragments into a new PCR plate, the specific steps include: controlling the island robotic arm to transfer the amplified multi-well plate to the pipetting workstation, and controlling the first pipette to transfer the amplification products in the multi-well plate into the first row of comb wells of the agarose gel electrophoresis apparatus.

[0093] The agarose gel electrophoresis apparatus is controlled to perform electrophoresis, and the target molecular weight band is separated under the monitoring system containing the imaging module until the target molecular weight band reaches the second row of comb wells of the agarose gel electrophoresis apparatus and the electrophoresis ends.

[0094] The first pipette is used to transfer the purified fragments from the second row of comb wells into a new PCR plate.

[0095] In some embodiments, when controlling the transfer of enzyme digestion system-related reagents into the PCR plate using a pipetting workstation to prepare the enzyme digestion system, the specific steps include the following:

[0096] The island robotic arm is controlled to transfer the PCR plate containing the purified fragment to the first metal bath at a first preset temperature, and to transfer other enzyme digestion system reagents stored below 0°C from a large-capacity light-proof high and low temperature chamber to the first metal bath.

[0097] The opening module is controlled to unscrew the caps of the reagent tubes corresponding to the enzyme digestion system; the first pipette is controlled to draw the reagents into the PCR plate; the opening module is controlled to reseal the caps of the reagent tubes; other required reagents are added to the PCR plate to prepare the enzyme digestion system; the PCR plate is then reacted in the first metal bath at a second preset temperature for 2-3 hours.

[0098] In some embodiments, the recovery of enzyme digestion products includes the following steps: recovering the enzyme digestion products using an agarose gel recovery method.

[0099] In some embodiments, constructing a ligation reaction system containing enzyme digestion products specifically includes the following steps: ice operation, controlling the island robotic arm and the first pipette to cooperate in adding the ligation reaction system to the PCR tube and mixing, and controlling the island robotic arm to move the PCR tube to the first metal bath at room temperature or to react overnight at room temperature for 8h~12h.

[0100] In some embodiments, when controlling the central robotic arm to deliver a new multi-well plate containing plasmids into the plasmid extraction and cell transformation plating workstation for transformation, the specific steps include the following:

[0101] The competent cell container is loaded onto the tray of the thermally conductive fixture in the cold metal bath mechanism. The second pipette is controlled to sequentially aspirate plasmids from the fed multi-well plate and add them to the competent cell container. The second plate-turning gripper is controlled to transfer the thermally conductive fixture and tray of the cold metal bath mechanism to the preheated hot metal bath mechanism for heat shock transformation for 45s~90s. During the transformation, the temperature and humidity-controlled automated incubator is controlled to eject the culture dish that has been pre-placed in the incubator through the transfer window. The central robotic arm is controlled to send the culture dish into the plasmid extraction and cell transformation plating workstation. The second plate-turning gripper is controlled to place the culture dish on the tray of the plating module and open the lid. The second plate-turning gripper is controlled to transfer the thermally conductive fixture and tray of the hot metal bath mechanism after heat shock transformation to the cold metal bath mechanism for cooling for 2min~10min.

[0102] In some embodiments, when transferring the transformed competent cells to the culture dish of the coating module, the specific steps include: controlling the second pipette to transfer the transformed competent cells to the culture dish of the coating module.

[0103] In some embodiments, while the plasmid extraction and cell transformation plating workstation is performing transformation, the pipetting workstation and peripheral equipment island are handling the next batch of samples.

[0104] In some embodiments, the cleaning and sterilization process includes the following steps: during cleaning, the spray tank is kept under negative pressure, and alkaline cleaning agent and pure water are sprayed and rinsed separately; during sterilization, infrared sterilization lamps are used to sterilize at 450℃~480℃.

[0105] The aforementioned molecular cloning device integrates a pipetting workstation and peripheral equipment island, a plasmid extraction and cell transformation plating workstation, a colony picker, a constant temperature and humidity automated incubator, and a central robotic arm into a single unit. This system achieves full automation of the entire process from DNA template to single-clone strain, enabling fully automated operation after manual sample loading. Experimenters do not need to intervene in the experiment except for the initial sample loading. The entire experimental process and process images are recorded and saved, reducing the workload of personnel while ensuring operational consistency and improving the automation level of the molecular cloning process. Because this application adopts fully automated operation, manual intervention in the experimental process is essentially unnecessary, reducing the risk of contamination introduced by operators and achieving the effect of reducing contamination introduction. Simultaneously, automated operation also improves operational efficiency.

[0106] Specifically, the aforementioned molecular cloning device features a closed operating environment. The pipetting workstation, PCR instrument, and sealing / tear-off device can all operate within this closed environment, reducing the possibility of airborne contaminants such as dust, microorganisms, and DNA / RNases entering the sample. The temperature- and humidity-controlled automated incubator maintains a stable, clean environment, preventing contamination of the strains or plasmids by other microorganisms. The central robotic arm and the island robotic arm work collaboratively to transfer samples and consumables, avoiding direct contact between laboratory personnel and samples, thus reducing the risk of human-induced contamination. The automated process is uniformly controlled by software, ensuring that each step conforms to standard procedures and avoiding contamination problems caused by human error, such as incorrect sample addition or disordered operation sequence. Furthermore, this application offers advantages in efficiency. The pipetting workstation supports multi-channel, variable-distance operation, enabling simultaneous processing of multiple samples, significantly improving liquid handling efficiency and providing high-throughput processing capabilities. The colony picker can quickly identify and select large numbers of colonies, far exceeding the speed and accuracy of manual plate picking. The central robotic arm automates the entire process from sample preparation, plasmid extraction, cell transformation, colony picking to culture, eliminating intermediate waiting time and shortening the overall experimental cycle. All sub-units coordinate their work through a unified control system, achieving "one-stop" process management. The molecular cloning device can operate continuously without human intervention, making it particularly suitable for experimental steps requiring long incubation or culture periods (such as overnight bacterial culture). During automated operation, all operation records are electronically stored, facilitating tracking and reproducibility of the experimental process. The automated process exhibits high consistency, contributing to stable and reliable experimental results and reducing the rate of repetitive experiments.

[0107] Furthermore, this application employs a functional zoning layout, specifically by setting up a pipetting workstation and peripheral equipment island, a plasmid extraction and cell transformation plating workstation, a colony picker, and a constant temperature and humidity automated incubator. This divides liquid handling, strain handling, picking, and culture operations into different devices based on functional zones and throughput levels. Each zone can operate independently or be connected as a whole to run the entire process. The four functional zones are connected in series via a central robotic arm, ensuring both process continuity and preventing mutual interference. DNA molecular-level processing is performed on traditional pipetting workstations and peripheral island equipment, without involving live cells. Processes requiring live colony handling, such as cell lysis for plasmid extraction, heat shock transformation, and culture dish plating, are performed on the plasmid extraction and cell transformation plating workstation. This workstation effectively integrates plasmid extraction, plasmid transformation into competent cells, and post-transformation cell plating. Compared to other manufacturers' solutions that use separate island equipment and robotic arms, this significantly improves processing efficiency by eliminating the need for the robotic arms to move between different devices. Colony picking, while simple, requires significant mechanical travel and is performed separately on the colony picker. Cell culture requires a stable culture environment and is performed separately in a temperature and humidity-controlled automated incubator. Attached Figure Description

[0108] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0109] To gain a more complete understanding of this application and its beneficial effects, the following description will be provided in conjunction with the accompanying drawings. In the following description, the same reference numerals denote the same parts.

[0110] Figure 1 This is a schematic diagram of the molecular cloning device described in one embodiment of this application;

[0111] Figure 2 This is a schematic diagram of the pipetting workstation and peripheral equipment island structure of the molecular cloning device described in one embodiment of this application;

[0112] Figure 3 This is a schematic diagram of the pipetting workstation and peripheral equipment island of the molecular cloning device described in one embodiment of this application from another angle;

[0113] Figure 4 This is a schematic diagram of the plasmid extraction and cell transformation plating workstation structure of the molecular cloning device described in one embodiment of this application;

[0114] Figures 5-8 This is a schematic diagram of the colony picking device of the molecular cloning apparatus described in one embodiment of this application.

[0115] Explanation of reference numerals in the attached figures

[0116] 1. Pipetting workstation and peripheral equipment island; 11. Pipetting workstation; 1101. Passive consumable plate holder base; 1102. Multi-station tilting stage; 1103. Trash can guide channel; 1104. First magnetic rack; 1105. First metal bath; 1106. Cap opening module; 1107. First pipette; 1108. First plate clamp; 1109. Agar gel electrophoresis apparatus; 1100. First heating and shaking module; 1110. Sample needle waste needle cleaning module; 1111. Sample needle 1112. Waste liquid needle; 1113. Vacuum suction cup; 12. Peripheral equipment island; 121. Screw cap test tube opener; 122. Large capacity light-shielding high and low temperature chamber; 123. Consumable tower; 124. Island robotic arm; 125. Transfer station; 13. Moving station; 2. Plasmid extraction and cell transformation plating workstation; 21. Second pipette; 22. Second plate clamp; 23. Second heating and shaking module; 24. Second magnetic rack; 25. Pipette tip holder; 26. Plasmid extraction Equipment; 261. Filter column; 262. Collection tube; 263. DNA extraction column; 264. Waste liquid tank; 265. Plasmid collection plate; 27. Trash can; 28. Cell transformation mechanism; 281. Cold metal bath mechanism; 282. Hot metal bath mechanism; 29. ​​Coating module; 3. Colony picker; 301. Culture dish inlet / outlet; 302. Culture medium inlet / outlet; 310. Feeding mechanism; 311. Extrusion mechanism; 3113. Driving wheel; 3114. Driven wheel; 312. Tube 320. Colony picking workbench; 330. Colony picking mechanism; 340. Cutting mechanism; 341. Cutting knife; 350. Drive mechanism; 361. Material; 362. Colony picking component; 4. Constant temperature and humidity automated incubator; 41. Transfer window; 5. Central robotic arm; 61. First guide component; 611. First guide rail; 612. Second guide rail; 613. First sliding seat; 62. Second guide component; 621. Third guide rail; 622. Fourth guide rail; 623. Second sliding seat. Detailed Implementation

[0117] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, the specific embodiments of this application are described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0118] In the description of this application, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc., indicating the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application.

[0119] In this application, unless otherwise expressly specified and limited, the terms "installation," "connection," "joining," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise expressly limited. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0120] In this application, unless otherwise expressly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "on top of," and "over" the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0121] In the description of this application, "several" means one or more, "multiple" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the stated number, while "above," "below," and "within" are understood to include the stated number. The use of "first" and "second" in the description is merely for distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the order of the indicated technical features.

[0122] In this document, "optionally," "optionally," and "optional" mean that something is optional, that is, it is selected from either "with" or "without." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent. In this application, descriptions such as "optionally contains" and "optionally includes" indicate "contains or does not contain."

[0123] In this document, unless otherwise stated, the reaction steps may be performed in the order described herein or not. For example, other steps may be included between reaction steps, and the order of reaction steps may be appropriately interchanged. This is something that those skilled in the art can determine based on conventional knowledge and experience. Preferably, the reaction methods described herein are performed sequentially.

[0124] In this application, when numerical intervals (i.e., numerical ranges) are mentioned, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed in this application should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include percentage intervals, ratio intervals, proportion intervals, etc.

[0125] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0126] This application provides a molecular cloning device to address at least one of the following technical problems in conventional molecular cloning: (1) During vector selection and construction, plasmid extraction generally relies on centrifuges and commercial reagent kits for column purification, which is cumbersome and difficult to integrate into automated processes. (2) After transformation of competent cells, manual coating is required, and sterilization and cleaning of the coating head require switching between multiple devices, increasing the risk of contamination. (3) After amplification and purification of the target gene, gel electrophoresis is generally used, requiring manual gel cutting and DNA recovery, which is time-consuming and prone to introducing impurities. (4) The pipetting workstations in conventional technologies have limited functions, and the workstations are mostly designed in a split manner, with each module operating independently, resulting in low module integration and reliance on manual intervention for sample transfer, leading to low efficiency. The molecular cloning device will be described below in conjunction with the accompanying drawings.

[0127] The molecular cloning apparatus provided in one embodiment of this application is exemplary; please refer to [link to relevant documentation]. Figure 1 As shown, Figure 1 This is a schematic diagram of a molecular cloning apparatus provided in one embodiment of this application. The molecular cloning apparatus of this application can improve the degree of automation, reduce the risk of contamination, and improve efficiency.

[0128] To illustrate the structure of the molecular cloning apparatus more clearly, the following description, in conjunction with the accompanying drawings, will be provided.

[0129] For example, please refer to Figure 1 As shown, a molecular cloning device includes a pipetting workstation 11, a peripheral equipment island 12, a mobile workstation 13, a PCR instrument, a plasmid extraction and cell transformation plating workstation 2, a colony picker 3, a constant temperature and humidity automated incubator 4, and a central robotic arm 5.

[0130] The pipetting workstation 11, peripheral equipment island 12, and mobile workstation 13 constitute the pipetting workstation and peripheral equipment island 1. Peripheral equipment island 12 is used for storing and transferring consumables, while mobile workstation 13 can interact with the central robotic arm 5 to transfer samples. Peripheral equipment island 12 includes an island robotic arm 124 for transferring reagents and consumables. Mobile workstation 13 is equipped with a sealing and tearing device and a centrifuge. Mobile workstation 13 can flexibly accommodate other equipment as needed for experiments, and can be connected to peripheral equipment island 12 before the experiment according to experimental requirements.

[0131] The PCR instrument is located at the pipetting workstation 11 or the mobile workstation 13.

[0132] The plasmid extraction and cell transformation plate-coating workstation 2 is used to extract plasmids and transform competent cells into plates.

[0133] The colony picker 3 is used to pick colonies from the petri dish into the liquid culture medium of the deep well plate.

[0134] The constant temperature and humidity automated incubator 4 is used for the automated culture of competent cells under constant temperature and humidity conditions.

[0135] The central robotic arm 5 is used to transfer samples and / or consumables between the moving station 13, the plasmid extraction and cell transformation plating workstation 2, the colony picker 3, and the constant temperature and humidity automated incubator 4.

[0136] The aforementioned molecular cloning device integrates a pipetting workstation and peripheral equipment island 1, a plasmid extraction and cell transformation plating workstation 2, a colony picker 3, a constant temperature and humidity automated incubator 4, and a central robotic arm 5 into a single unit. This system achieves full automation of the entire process from DNA template to single-clone strain, enabling fully automated operation after manual sample loading. Experimenters do not need to intervene in the experiment except for the initial sample loading. The entire experimental process and process images are recorded and saved, reducing the workload of personnel while ensuring operational consistency, improving the automation level of the molecular cloning process, reducing contamination introduction, and increasing efficiency.

[0137] In some embodiments, the colony picker 3 may be a commercially available colony picker. For example, the colony picker 3 may be a commercially available QuickPick 300 model manufactured by Measuring Instruments.

[0138] In some embodiments, the temperature and humidity automated incubator 4 can be a commercially available temperature and humidity automated incubator. For example, the model of the temperature and humidity automated incubator 4 is the commercially available Xinhua Medical BCC-80A model.

[0139] In some implementations, see Figure 2 , Figure 3 As shown, Figure 2 This is a schematic diagram of the pipetting workstation and peripheral equipment island structure of a molecular cloning device according to an embodiment of this application. Figure 3 This is a schematic diagram of the pipetting workstation and peripheral equipment island of a molecular cloning device according to an embodiment of this application from another angle. The pipetting workstation 11 includes a pipetting worktable and connected to the pipetting worktable: a first magnetic rack 1104 for adsorbing magnetic beads, a first metal bath 1105 for isothermal preservation of reagents at a temperature of 4℃~60℃, a capping module 1106 for opening or resealing cryovials, a first pipette 1107 for pipetting, a first rotating plate gripper 1108 for transferring container consumables or consumable trays between the moving workstations 13 of the pipetting workstation, a PCR product separation and purification device for separating and purifying PCR products, a first heating and shaking module 1100 for heating and rotating well plates for mixing, a sample dispensing needle 1111 for adding reagents such as buffer solutions and connecting to a multi-channel rotary valve, a waste liquid needle 1112 for removing waste liquid and connecting to a peristaltic pump, and a vacuum suction cup 1113 for opening the petri dish lid.

[0140] In some embodiments, the first rotary plate gripper 1108 includes an openable gripper, a gripper opening and closing drive component, and a rotary plate gripper drive component. The gripper drive component is mounted on the pipetting stage. The gripper drive component is connected to the gripper opening and closing drive component to drive the gripper and the gripper to move. The gripper opening and closing drive component is connected to the gripper to drive the gripper to open and close.

[0141] In some embodiments, the first metal bath 1105 may be a dual-station metal bath 1105.

[0142] In some embodiments, the pipetting workstation 11 also includes a passive consumables tray base 1101 connected to the pipetting worktable for storing consumables that do not require power, such as pipette tip boxes and room temperature reagents.

[0143] In some embodiments, the pipetting station 11 also includes a multi-station tilting stage 1102 connected to the pipetting stage for tilting the orifice plate to concentrate the liquid to one side edge.

[0144] In some embodiments, the multi-station tilting stage 1102 is connected to a tilting stage drive component. The multi-station tilting stage 1102 is rotatably connected to the pipetting stage. Under the drive of the tilting stage drive component, the multi-station tilting stage 1102 can rotate to the required tilting angle and maintain it. The tilting stage drive component can be a drive motor or a lead screw assembly.

[0145] In some embodiments, the pipetting workstation 11 also includes a trash can guide 1103 connected to the pipetting worktable for accommodating and storing waste consumables 27.

[0146] In some embodiments, the PCR product separation and purification apparatus includes an agarose gel electrophoresis apparatus 1109. The agarose gel electrophoresis apparatus 1109 has double rows of comb wells to allow for visual guidance of a first pipette 1107 to directly aspirate the target molecular weight band from the second row of wells of the agarose gel electrophoresis apparatus 1109 when electrophoresis reaches the second row of comb wells.

[0147] It is easy to understand that in other examples, PCR product separation and purification devices also include magnetic bead purification devices. The core principle of magnetic bead purification is that, under the conditions of high salt and polyethylene glycol (PEG), the groups modified on the surface of the magnetic beads will specifically adsorb DNA molecules. By changing the salt concentration and other conditions of the solution, the binding and elution of DNA with the magnetic beads can be reversibly controlled, thereby achieving the purification and concentration of PCR products.

[0148] In some embodiments, the pipetting workstation 11 further includes a sample needle waste liquid cleaning module 1110 connected to the pipetting worktable for cleaning sample needles and / or waste liquid needles.

[0149] In some embodiments, the pipetting stage is further provided with a first guide component capable of moving laterally and longitudinally in the horizontal direction, and the capping module 1106, the first pipetting gun 1107, the first rotating plate gripper 1108, the sample needle 1111, and the waste liquid needle 1112 are respectively disposed on the first guide component.

[0150] In some embodiments, the first guide component includes a first guide rail extending laterally in the horizontal direction, a second guide rail slidably connected to the first guide rail, and a first sliding seat slidably connected to the second guide rail. The cap opening module 1106, the first pipette 1107, the first rotating plate gripper 1108, the sample dispensing needle 1111, and the waste liquid needle 1112 are respectively disposed on the first sliding seat.

[0151] In some embodiments, the capping module 1106 independently rotates to open the cap of a single-channel cryopreservation tube along the Z-axis. The capping module 1106 is capable of moving independently in the vertical direction and can open or close the cap of one cryopreservation tube at a time.

[0152] In some embodiments, the cap-opening module 1106 includes a first cap-opening gripper, a second cap-opening gripper, a first cap-opening drive component, and a second cap-opening drive component. The first and second cap-opening drive components are respectively mounted on a pipetting stage. The first cap-opening gripper is connected to the first cap-opening drive component, which drives the first cap-opening gripper to move and grip the test tube. The second cap-opening drive component is connected to the second cap-opening gripper to drive the second cap-opening gripper to move and grip the test tube cap. The second cap-opening drive component drives the second cap-opening gripper to move in the opposite direction to open or close the test tube cap. The aforementioned first and second cap-opening drive components can be drive motors or lead screw assemblies.

[0153] In some embodiments, the first pipette 1107 includes a first multi-channel variable-pitch independent Z-axis pipette. The first pipette 1107 can control each pipette tip to move independently in the vertical direction and can adjust the distance between different channels as needed to accommodate multi-well plates of different sizes.

[0154] In some embodiments, the sampling needle 1111 includes an independent Z-axis sampling needle. The sampling needle 1111 is capable of moving independently in the vertical direction.

[0155] In some embodiments, the waste syringe 1112 includes an independent Z-axis waste syringe. The sample dispensing syringe 1112 is capable of moving independently in the vertical direction.

[0156] In some embodiments, the peripheral equipment island 12 also includes a screw-on test tube opener 121 for opening test tubes, a large-capacity light-shielding high-low temperature chamber 122 for incubating samples in well plates and temporarily storing low-temperature reagents, a consumable tower 123 for storing consumables including pipette tip boxes and well plates, and a transfer station 125 for interacting with the central robotic arm 5 to exchange samples. The island robotic arm 124 can obtain reagent sample containers or trays containing reagent consumable containers from the screw-on test tube opener 121 and the large-capacity light-shielding high-low temperature chamber 122 and transfer them to the transfer station 125.

[0157] In some embodiments, the peripheral device island 12 is located downstream of the pipetting workstation 11.

[0158] In some embodiments, the peripheral equipment island 12 also includes an island workbench, and an island robotic arm 124 is slidably connected to the island robotic arm 124 to transfer reagents and consumables between the large-capacity light-shielding high and low temperature chamber 122 and the consumable tower 123.

[0159] In some embodiments, the screw capping test tube opener 121 can be selected from the Z-Arm2442 four-axis robotic arm, Z-EFG-20 bottom gripper, and Z-ERG-20C rotary capping gripper manufactured by Huiling Technology Co., Ltd.

[0160] In some embodiments, the mobile workstation 13 includes multiple units. Each mobile workstation 13 is equipped with one of the following: a PCR instrument, a sealing and tearing device, and a centrifuge. For example, there are three mobile workstations 13.

[0161] In some of these implementations, please refer to Figure 4 As shown, Figure 4 The plasmid extraction and cell transformation plating workstation structure of a molecular cloning device according to an embodiment of this application includes a second pipette 21 for pipetting, a second plate clamp 22 for gripping and moving a transfer fixture containing competent cell tubes, a second heating and oscillating module 23 for heating and oscillating the well plate, a second magnetic rack 24 for adsorbing magnetic beads, a plasmid extraction device 26 for automatically extracting plasmids, a cell transformation mechanism 28 for transforming competent cells, and a plating module 29 for coating competent cells.

[0162] In some embodiments, the first heating and oscillation module 1100 and the second heating and oscillation module 23 are independently selected from instruments that integrate heating (temperature control) and oscillation (shaking / mixing) functions. The instrument includes a heating function: providing a stable and precise temperature environment for the sample through a precision electric heating system and temperature sensors; and an oscillation function: using a motor-driven platform, causing sample containers (such as test tubes, centrifuge tubes, multi-well plates, etc.) placed on it to reciprocate or circularly move at a specific speed, amplitude, and trajectory, thereby achieving the purpose of mixing, oscillation, or incubation.

[0163] In some embodiments, the plasmid extraction apparatus 26 includes: a filter column 261 for filtering cell lysate; a collection tube 262 located below the filter column 261 for collecting plasmids filtered by the filter column 261 and purifying the plasmids with magnetic beads; a DNA extraction column 263 for enriching plasmids from the collection tube 262 onto a silica membrane after positive pressure; a waste liquid tank 264 for collecting waste liquid from the filter column 261; and a plasmid collection plate 265. The plasmid collection plate 265 is used to collect plasmids purified by magnetic beads from the collection tube 262, or to collect plasmids from the silica membrane of the DNA extraction column 263.

[0164] In some embodiments, plasmid extraction device 26 employs a positive pressure column plasmid extraction device.

[0165] In some embodiments, the plasmid extraction device 26, during operation, includes the following steps: lysate from cell lysis is added to a filter column 261; a positive pressure module above the filter column 261 presses down to seal the filter column 261 and provides positive pressure; the sample is filtered into a collection tube 262 below the filter column 261; magnetic beads are then added to the collection tube 262; the liquid in the collection tube 262 is purified by the magnetic attraction of the first magnetic frame 1104; after magnetic bead purification, the plasmid is collected; or, a DNA extraction column 263 is used to pass the sample through a positive pressure test again. Waste liquid is collected in a waste tank 264; the DNA extraction column 263 is transferred to a plasmid collection plate 265; the plasmids enriched on the silica membrane of the DNA extraction column 263 are eluted back onto the plasmid collection plate 265 and collected. The discarded filter column 261 is collected in a trash can 27.

[0166] In some embodiments, the cell transformation mechanism 28 includes a second metal bath for applying a cold or hot bath to the competent cell tubes held in place on the transfer fixture.

[0167] In some embodiments, the second metal bath includes a cold metal bath mechanism 281 and a hot metal bath mechanism 282. The cold metal bath mechanism 281 is used to cold bath the competent cell tube stuck on the transfer fixture. The hot metal bath mechanism 282 is used to hot bath the competent cell tube stuck on the transfer fixture. The cold metal bath mechanism 281 and the hot metal bath mechanism 282 alternately cold bath and hot bath the competent cell tube, respectively.

[0168] In some of these embodiments, the cold metal bath mechanism 281 has multiple channel stations, enabling it to be matched to the 96-well throughput of the plasmid collection plate.

[0169] In some embodiments, the second pipette 21 includes a second multi-channel variable-pitch independent Z-axis pipette. The second pipette 21 can control the independent movement of each pipette tip in the vertical direction and can adjust the distance between different channels as needed to accommodate multi-well plates of different sizes.

[0170] In some embodiments, the extraction conversion coating worktable is further provided with a second guide component capable of moving laterally and longitudinally in the horizontal direction, and the second pipette 21 and the second rotating plate gripper 22 are respectively disposed on the second guide component.

[0171] In some embodiments, the second guide component includes a third guide rail extending laterally in the horizontal direction, a fourth guide rail slidably connected to the third guide rail, and a second sliding seat slidably connected to the fourth guide rail, with the second pipette 21 and the second rotating plate gripper 22 respectively disposed on the second sliding seat.

[0172] In some embodiments, the coating module 29 includes a lifting and rotating coating head. The lifting and rotating coating head is mounted on an indexing plate, which can rotate to move the coating head to a cleaning station, a high-temperature infrared sterilization station, and a drying station, respectively for cleaning, high-temperature infrared sterilization, and drying. It should be noted that the aforementioned lifting and rotating coating head refers to a coating head that can move both linearly in the Z-direction and rotate in the R-direction.

[0173] In some embodiments, the plasmid extraction and cell transformation plating workstation 2 also includes a pipette tip holder 25 for storing pipette tip holders.

[0174] In some embodiments, the plasmid extraction and cell transformation plating workstation 2 also includes a trash can 27 for storing waste consumables and waste filter columns.

[0175] In some embodiments, the colony picker 3 has a petri dish inlet / outlet 301 and a culture medium inlet / outlet 302. The petri dish inlet / outlet 301 is used to interact with the central robotic arm 5 to pick up the petri dish containing the colonies; the culture medium inlet / outlet 302 is used to interact with the central robotic arm 5 to store the picked colonies in a culture medium container.

[0176] In some implementations, see Figures 5-8 As shown, Figures 5-8 This is a schematic diagram of the colony picker structure of a molecular cloning device according to an embodiment of this application. The colony picker 3 includes a colony picking workbench 320, a feeding mechanism 310, a picking mechanism 330, a driving mechanism 350, and a cutting mechanism 340.

[0177] The colony picking workbench 320 is used to hold several petri dishes.

[0178] The feeding mechanism 310 is installed on the colony picking workbench 320 and is used to continuously output material 361.

[0179] The colony picking mechanism 330 is positioned above the colony picking workbench 320. The colony picking mechanism 330 is used to receive the material 361 output by the feeding mechanism 310 and drive the head end of the material 361 to output a preset length in the direction close to the colony picking workbench 320 to form a colony picking piece 362 for picking colonies.

[0180] The drive mechanism 350 is connected to the colony-picking mechanism 330. The drive mechanism 350 is used to drive the colony-picking mechanism 330 to move so that the colony-picking mechanism 330 drives the colony-picking element 362 to pick up colonies from one of the petri dishes, and / or, to drive the colony-picking mechanism 330 to drive the colony-picking element 362 to plant colonies in another petri dish.

[0181] The cutting mechanism 340 is used to separate the picker 362 from the material 361.

[0182] In some embodiments, the feeding mechanism 310 includes a conduit 312 and an extrusion mechanism 311.

[0183] One end of the conduit 312 is connected to the bacteria-picking mechanism 330;

[0184] The extrusion mechanism 311 is mounted on the colony picking workbench 320 and connected to the other end of the conduit 312. The extrusion mechanism 311 is used to transport the material 361 along the conduit 312 to the colony picking mechanism 330.

[0185] In some implementations, see Figure 8As shown, the extrusion mechanism 311 includes a drive wheel 3113 rotatably mounted on the colony picking worktable 320 and a driven wheel 3114 spaced apart from the drive wheel 3113. A conveying channel for conveying material 361 is formed between the driven wheel 3114 and the drive wheel 3113, and the drive wheel 3113 can be driven to rotate by power. Driving the drive wheel 3113 enables the drive wheel 3113 and the driven wheel 3114 to cooperate in driving the material 361 forward.

[0186] In some embodiments, the cutting mechanism 340 includes a cutting blade 341. The cutting blade 341 is movably disposed on the colony picking worktable 320. The cutting blade 341 is actuable to cut off the material 361 at the end of the conduit 312.

[0187] In some embodiments, the colony picker 3 also includes a metering component for measuring the length of material 361 conveyed each time. When the length of material 361 conveyed reaches a preset length, the drive wheel 3113 stops rotating.

[0188] One embodiment of this application also provides a molecular cloning method.

[0189] A molecular cloning method, employing the molecular cloning apparatus of any of the above embodiments, includes the following steps:

[0190] S10. Control the island robotic arm 124 to transfer the multi-well plate from the sub-zero temperature environment of the peripheral equipment island 12 to the centrifuge in the moving station 13, and control the centrifuge to centrifuge; control the island robotic arm 124 to transfer the multi-well plate to the sealing and tearing device in the moving station 13, control the sealing and tearing device to remove the sealing film on the multi-well plate, control the island robotic arm 124 to transfer the multi-well plate in the sealing and tearing device to the first metal bath 1105 in the pipetting workstation 11 with the temperature set to the first preset temperature; control the island robotic arm 124 to transfer the low-temperature reagent in the peripheral equipment island 12 to the first metal bath 1105 in the pipetting workstation 11. A metal bath 1105; control the pipetting workstation 11 to transfer the required low-temperature reagents into the wells of the multi-well plate, and add other required reagents to prepare the PCR reaction system; control the island robotic arm 124 to send the multi-well plate into the PCR instrument on the moving station 13, control the PCR instrument to amplify, and after amplification, the multi-station tilting stage 1102 can be used to tilt the plate so that the liquid in the multi-well plate is concentrated on one side edge, which is convenient for aspiration and transfer; the PCR amplification product is separated and purified by the agar gel electrophoresis instrument 1109 of the pipetting workstation 11, and the purified fragment is transferred to a new PCR well plate.

[0191] S20. Control the island robotic arm 124 to transfer the PCR plate containing the purified fragment to the first metal bath 1105 with the temperature set to the first preset temperature. Control the island robotic arm 124 to transfer other enzyme digestion system related reagents stored below 0℃ from the peripheral device island 12 to the first metal bath 1105. Control the pipetting workstation 11 to transfer enzyme digestion system related reagents into the PCR plate to prepare the enzyme digestion system, and react in the first metal bath 1105 with the temperature set to the second preset temperature for 2h~3h. Recover the enzyme digestion products. Construct a ligation reaction system containing the enzyme digestion products and mix it in the PCR tube. React overnight at room temperature for 8h~12h to obtain the plasmid that integrates the target DNA fragment into the vector plasmid. Extract the plasmid.

[0192] S30. Control the central robotic arm 5 to send the multi-well plate containing plasmids into the plasmid extraction and cell transformation coating workstation 2 for transformation, and transfer the transformed competent cells to the culture dish of the coating module 29; control the coating module 29 to spread the transformed competent cells evenly, and perform cleaning and sterilization procedures.

[0193] S40, the central robotic arm 5 and the island robotic arm 124 work together to invert the coated culture dish and transfer it to the constant temperature and humidity automated incubator 4 for cultivation.

[0194] S50. After the culture is completed, the central robotic arm 5 sequentially sends the culture dishes into the colony picker 3 for colony selection. The picked colonies are stored in a deep-well plate containing culture medium.

[0195] S60. Colony selection is performed using colony selection, specifically including the following steps: The central robotic arm 5 sequentially feeds the culture dish into the colony picking table 320 of the colony picking instrument 3. The feeding mechanism 310 continuously outputs material 361 (material 361 can be a filamentous thermoplastic material or metal wire). Material 361 is conveyed through the conveying channel formed between the driven wheel 3114 and the driving wheel 3113. The extrusion mechanism 311 conveys material 361 along the guide tube 312 to the picking mechanism 330. The metering component measures the length of material 361 conveyed each time. When the length of material 361 conveyed reaches the preset length, the driving wheel 3113 stops rotating. The cutting blade 341 of the cutting mechanism 340 is moved to a predetermined position and actuated to cut the material 361 at the end of the guide tube 312. The head end of material 361 is output in the direction close to the colony picking table 320 for a preset length to form a picking piece 362 for picking bacteria. The control drive mechanism 350 drives the picking mechanism 330 to move, so that the picking mechanism 330 drives the picking element 362 to pick up colonies from one of the petri dishes, and drives the picking mechanism 330 to drive the picking element 362 to plant the colonies in another petri dish.

[0196] In some embodiments, the first preset temperature includes 4°C to 60°C. Further, the first preset temperature includes 4°C to 25°C.

[0197] In some embodiments, the second preset temperature includes 25°C to 60°C. Further, the second preset temperature includes 30°C to 37°C.

[0198] In some embodiments, before the robotic arm 124 controls the transfer of the multiwell plate from the sub-zero temperature environment of the peripheral equipment island 12 to the centrifuge, the following preparatory work is included: placing the multiwell plate containing DNA template and sealed with a membrane, along with low-temperature reagents such as primers, into a large-capacity, light-shielded high-low temperature chamber 122; opening and resealing the caps of the low-temperature reagent tubes in the large-capacity, light-shielded high-low temperature chamber 122 using a screw-on tube opener 121; placing room-temperature reagents, pipette tip boxes, and other consumable containers into designated positions on the passive consumables plate base 1101; and configuring a PCR instrument, a membrane sealing and tearing device, and a centrifuge at the mobile workstation 13.

[0199] In some embodiments, when the island robotic arm 124 is controlled to transfer the perforated plate from the sub-zero temperature environment of the peripheral equipment island 12 to the centrifuge of the moving station 13, the specific steps include: controlling the island robotic arm 124 to transfer the perforated plate from the sub-zero temperature environment in the large-capacity light-shielding high and low temperature chamber 122 of the peripheral equipment island 12 to the centrifuge of the moving station 13.

[0200] In some embodiments, the control island robotic arm 124 picks up a balancing orifice plate from the consumable tower 123 for balancing the centrifuge.

[0201] In some embodiments, when the control pipetting station 11 transfers the required cryogenic reagent into the well of the multi-well plate, the following steps are included: controlling the cap opening module 1106 to unscrew the cap of the cryogenic reagent tube, controlling the first pipette 1107 to draw the required cryogenic reagent into the well of the multi-well plate, and controlling the cap opening module 1106 to reseal the cap.

[0202] In some embodiments, when adding other required reagents to the wells of the multi-well plate to prepare the PCR reaction system, the following steps are included: controlling the first pipette 1107 to add other required reagents to prepare the PCR reaction system. It should be noted that the aforementioned other required reagents refer to the essential reagents for preparing the PCR reaction system.

[0203] In some embodiments, when the PCR amplification products are separated and purified by the agarose gel electrophoresis apparatus 1109 of the pipetting workstation 11 and the purified fragments are transferred to a new PCR plate, the specific steps include the following: controlling the island robotic arm 124 to transfer the amplified multi-well plate to the pipetting workstation 11, and controlling the first pipette 1107 to transfer the amplification products in the multi-well plate to the first row of comb wells of the agarose gel electrophoresis apparatus 1109.

[0204] Electrophoresis is performed by controlling the agarose gel electrophoresis apparatus 1109. The target molecular weight bands are separated under the monitoring system containing the imaging module until the target molecular weight bands reach the second row of comb wells of the agarose gel electrophoresis apparatus 1109 and the electrophoresis ends.

[0205] Control the first pipette 1107 to transfer the purified fragments in the second row of comb wells into a new PCR plate.

[0206] In some embodiments, plasmid extraction specifically includes the following steps: using plasmid extraction device 26. Cell lysate is added to filter column 261. A positive pressure module above filter column 261 presses down to seal the filter column 261 and provides positive pressure, filtering the sample into collection tube 262 below filter column 261. Magnetic beads are then added to collection tube 262, and the liquid in collection tube 262 is purified by adsorption of the magnetic beads under the magnetic attraction of the first magnetic frame 1104. After magnetic bead purification, the plasmid is collected. Waste filter column 261 is collected in trash can 27.

[0207] In some embodiments, when controlling the pipetting workstation 11 to transfer the enzyme digestion system-related reagents into the PCR well plate to prepare the enzyme digestion system, the specific steps include the following:

[0208] The island robotic arm 124 is controlled to transfer PCR plates containing purified fragments to a first metal bath 1105 with the temperature set to a first preset temperature. The island robotic arm 124 is also controlled to transfer other enzyme digestion system related reagents, such as low-temperature enzyme reagents, stored below 0°C, from a large-capacity light-proof high and low temperature chamber 122 to the first metal bath 1105.

[0209] The opening module 1106 is controlled to unscrew the caps of the corresponding enzyme digestion system reagents, and the first pipette 1107 is controlled to aspirate the enzyme digestion system reagents into the PCR plate. The opening module 1106 is then controlled to reseal the caps of the enzyme digestion system reagents. The first pipette 1107 is then controlled to continue adding other required reagents into the PCR plate to prepare the enzyme digestion system. The PCR plate is then controlled to react in the first metal bath 1105, which is set to the second preset temperature, for 2-3 hours.

[0210] In some embodiments, the recovery of enzyme digestion products includes the following steps: recovering the enzyme digestion products using an agarose gel recovery method.

[0211] In some embodiments, the construction of a ligation reaction system containing enzyme digestion products specifically includes the following steps: ice operation, controlling the island robotic arm 124 and the first pipette 1107 to add the ligation reaction system to the PCR tube and mix well, controlling the island robotic arm 124 to move the PCR tube to the first metal bath 1105 with the temperature set to room temperature or to react overnight at room temperature for 8h~12h.

[0212] In some embodiments, before the central robotic arm 5 delivers the multi-well plate containing plasmids into the plasmid extraction and cell transformation plating workstation 2 for transformation, a pipette tip box is mounted on the pipette tip box holder 25 so that the second pipette 21 can obtain the tip.

[0213] In some embodiments, when controlling the central robotic arm 5 to deliver a multi-well plate containing plasmids into the plasmid extraction and cell transformation plating workstation 2 for transformation, the specific steps include the following:

[0214] The competent cell containers are manually loaded onto the tray of the thermally conductive fixture in the cold metal bath mechanism 281 beforehand. The second pipette 21 is controlled to sequentially aspirate plasmids from the fed multi-well plate and add them to the competent cell containers. The second plate-turning gripper 22 is controlled to transfer the thermally conductive fixture and tray of the cold metal bath mechanism 281 to the preheated hot metal bath mechanism 282 for thermal shock conversion for 45s~90s. During the conversion, the temperature and humidity constant automatic incubator 4 is controlled to pop out the culture dish that has been pre-placed in the incubator through the transfer window 41. The lid of the culture dish is opened and resealed by the vacuum suction cup 1113. The central robotic arm 5 is controlled to send the culture dish into the plasmid extraction and cell transformation coating workstation 2. The second plate-turning gripper 22 is controlled to place the culture dish on the tray of the coating module 29 and open the lid through the vacuum suction cup 1113. The second plate-turning gripper 22 is controlled to transfer the thermally conductive fixture and tray in the hot metal bath mechanism 282 after thermal shock conversion to the cold metal bath mechanism 281 for cooling for 2min~10min.

[0215] In some embodiments, when transferring the transformed competent cells to the culture dish of the coating module 29, the specific steps include: controlling the second pipette 21 to transfer the transformed competent cells to the culture dish of the coating module 29, and the lid of the culture dish is opened or closed by the vacuum suction cup 1113.

[0216] In some embodiments, while plasmid extraction and cell transformation plating workstation 2 is performing transformation, pipetting workstation and peripheral equipment island 1 are operating on the next batch of samples.

[0217] In some embodiments, the cleaning and sterilization process includes the following steps: during cleaning, the spray tank is kept under negative pressure, and alkaline cleaning agent and pure water are sprayed and rinsed separately; during sterilization, infrared sterilization lamps are used to sterilize at 450℃~480℃.

[0218] In some embodiments, the alkaline cleaning agent includes Decon90.

[0219] In some embodiments, the perforated plate described above can be a 96-well plate, and the following specific examples will use a 96-well plate for illustration.

[0220] Implementation Method 1

[0221] This embodiment provides a molecular cloning method. The molecular cloning method includes the following steps:

[0222] (1) Place the 96-well plate containing DNA template and sealed with a membrane, and low-temperature reagents such as primers into a large-capacity light-shielding high and low temperature chamber 122. Place room-temperature reagents, pipette tip boxes, and container consumables into the designated positions on the passive consumable plate base 1101. Configure a PCR instrument, a sealing and tearing device, and a centrifuge at the mobile work station 13.

[0223] (2) Control the island robotic arm 124 to transfer the 96-well plate from the sub-zero temperature environment in the large-capacity light-proof high and low temperature chamber 122 to the centrifuge at the moving station 13. Control the island robotic arm 124 to pick up the balancing plate from the consumable tower 123 for balancing the centrifuge. Control the centrifuge to centrifuge the DNA template solution to the bottom of the 96-well plate for automated operation.

[0224] (3) After centrifugation, control the island robotic arm 124 to transfer the 96-well plate to the sealing and tearing device of the moving station 13, control the sealing and tearing device to remove the sealing film on the 96-well plate, and control the island robotic arm 124 to transfer the 96-well plate in the sealing and tearing device to the first station of the first metal bath 1105 in the liquid transfer station 11 with the temperature set to the first preset temperature.

[0225] (4) Control the island robotic arm 124 to transfer the low-temperature (stored below 0°C) reagents, such as enzyme reagents, from the large-capacity light-shielding high and low temperature chamber 122, along with the matching fixtures, to the second station of the first metal bath 1105.

[0226] (5) Control the cap opening module 1106 to unscrew the cap of the low temperature reagent, control the first pipette 1107 to draw the required low temperature reagent into the well of the 96-well plate, and control the cap opening module 1106 to reseal the cap.

[0227] (6) Control the first pipette 1107 to add other required reagents to prepare the PCR reaction system, control the island robotic arm 124 to send the 96-well plate into the PCR instrument on the moving station 13, and control the PCR instrument to amplify.

[0228] (7) Control the island robotic arm 124 to transfer the amplified 96-well plate to the pipetting workstation 11, and control the first pipette 1107 to transfer the amplification product in the 96-well plate to the first row of comb wells of the agar gel electrophoresis apparatus 1109.

[0229] Electrophoresis is performed by controlling the agarose gel electrophoresis apparatus 1109. The target molecular weight bands are separated under the monitoring system containing the imaging module until the target molecular weight bands reach the second row of comb wells of the agarose gel electrophoresis apparatus 1109 and the electrophoresis ends.

[0230] The purified fragments in the second row of comb wells were transferred to a new PCR plate using the first pipette 1107 for subsequent enzyme digestion and ligation. This application used a double-comb agarose gel electrophoresis apparatus 1109 for purification, separating nucleic acid fragments of specified molecular weights, thus improving the continuity of downstream experiments and the reliability of sample purity.

[0231] (8) Enzyme digestion and ligation

[0232] (8-1) Constructing the enzyme digestion system

[0233] The robotic arm 124 on the control island transfers the PCR plate containing the purified fragment to the first station of the first metal bath 1105, which is set to the first preset temperature. The robotic arm 124 on the control island transfers other enzyme digestion system related reagents stored below 0°C, such as low-temperature enzyme reagents, together with the matching fixtures, from the large-capacity light-proof high and low temperature chamber 122 to the second station of the first metal bath 1105. The cap opening module 1106 is controlled to unscrew the caps of the corresponding enzyme digestion system related reagents. The first pipette 1107 is controlled to draw the enzyme digestion system related reagents into the PCR plate. The cap opening module 1106 is controlled to reseal the caps of the enzyme digestion system related reagents. The first pipette 1107 is controlled to continue adding other required reagents into the PCR plate to prepare the enzyme digestion system. The PCR plate is controlled to react in the first metal bath 1105, which is set to the second preset temperature, for 2 to 3 hours.

[0234] (8-2) The enzyme digestion products were recovered using the agarose gel recovery method in step (7) above.

[0235] (8-3) Establishing a connecting reaction system

[0236] The process is carried out on ice. The island robotic arm 124 and the first pipette 1107 work together to add the ligation reaction system into the PCR tube and mix it. The island robotic arm 124 is then controlled to move the PCR tube to the first station of the first metal bath 1105, which is set to room temperature, or to react overnight at room temperature for 8 to 12 hours to obtain a plasmid that integrates the target DNA fragment into the vector plasmid.

[0237] (8-4) Transformation

[0238] The island robotic arm 124 is controlled to place the 96-well plate containing the plasmid from step (8-3) into the transfer station 125, and the central robotic arm 5 is controlled to send the 96-well plate containing the plasmid into the plasmid extraction and cell transformation plating workstation 2.

[0239] At this time, the pipetting workstation and peripheral equipment island 1 are completely idle and can be used to process the next batch of samples. By integrating the continuous process flow into a set of equipment, the overall efficiency of the entire device can be improved, avoiding the inefficiency of other manufacturers' solutions where only one island robotic arm 124 transfers consumables between multiple independent workstations.

[0240] The competent cell containers are pre-loaded manually onto the tray of the thermally conductive fixture in the cold metal bath mechanism 281. The second pipette 21 is then used to sequentially aspirate plasmids from the 96-well plate and add them to the competent cell containers. Since each group of cold metal bath mechanisms 281 consists of 4×6 rows of stations, totaling 4 cold metal bath mechanisms 281, the above operation is performed in 4 groups. Mixing is performed by pipetting if necessary. Other combinations of numbers can be used, or standard SBS rectangular multi-well plates can be used instead of individual containers. The second rotating plate gripper 22 is used to transfer the thermally conductive fixture and tray of the cold metal bath mechanism 281 to the preheated hot metal bath mechanism 282 for 4... The heat shock conversion lasts from 5s to 90s. During the conversion process, the temperature and humidity controlled automated incubator 4 ejects the culture dish that has been pre-placed in the incubator through the transfer window 41. The central robotic arm 5 sends the culture dish into the plasmid extraction and cell transformation plating workstation 2. The second plate-turning gripper 22 places the culture dish on the tray of the plating module 29 and opens the lid. The second plate-turning gripper 22 transfers the heat-conducting fixture and tray that have completed the heat shock conversion in the hot metal bath mechanism 282 to the cold metal bath mechanism 281 for cooling for 2min to 10min. The second pipette 21 transfers the transformed competent cells into the culture dish of the plating module 29.

[0241] (9) Control the coating module 29 to coat the transformed competent cells evenly and perform cleaning and sterilization processes; during cleaning, the spray tank is kept under negative pressure and alkaline cleaning agent and pure water are used for spraying and rinsing respectively; sterilization is carried out by infrared sterilization lamp at 450℃.

[0242] (10) Control the second rotating plate gripper 22 to transfer the coated culture dish to the transfer window 41, control the central robotic arm 5 and the island robotic arm 124 to reverse the coated culture dish and transfer it to the constant temperature and humidity automated incubator 4 through the transfer window 41 for culture.

[0243] (11) After the culture is completed, the central robotic arm 5 will send the culture dishes into the culture dish inlet 311 of the colony picker 3 in sequence for colony selection. The picked colonies are stored in the deep well plate containing culture medium at the culture medium inlet 312.

[0244] Implementation Method 2

[0245] This embodiment describes the process from plasmid amplification of monoclonal strain culture to protein expression monoclonal strain.

[0246] The 96-well plate delivered from the culture medium inlet / outlet 312 is placed into the transfer station 125 by the central robotic arm 5. The island robotic arm 124 seals the plate with a breathable membrane on the sealing and tearing device and then sends it into the first heating and shaking module 1100 of the pipetting workstation 11 for temperature-controlled shaking incubation. When the bacteria in each well of the 96-well plate have multiplied to a certain amount, the culture medium is separated by centrifugation in a centrifuge. Then the membrane is torn off in the sealing and tearing device, and the supernatant is aspirated in the pipetting workstation 11 and transferred again into the plasmid extraction and cell transformation plating workstation 2. The plate is then placed on the second heating and shaking module 23 by the second plate transfer gripper 22.

[0247] Control the second pipette 21 to add the resuspension, lysis buffer, neutralization buffer, and binding buffer to the wells of each 96-well plate, and perform mixing and shaking operations in between to complete the cell lysis and lysis buffer preparation.

[0248] The second pipette 21 is controlled to add cell lysis buffer to the 96-well filter column 261. The tray containing the filter column 261 is moved under the positive pressure module. The upper positive pressure module is pressed down and sealed to provide positive atmospheric pressure to filter the sample into the collection tube 262 below. The second rotating plate gripper 22 discards the filter column 261 into the trash can 27.

[0249] a. Add magnetic beads to bind DNA to the magnetic beads in the collection tube 262, and then remove the waste liquid and elute the plasmid on the second magnetic rack 24.

[0250] b. Add the liquid in collection tube 262 to DNA extraction column 263 using a pipette and pass it under positive pressure to collect the waste liquid in waste liquid tank 264. Then transfer DNA extraction column 263 to the plasmid collection plate 265 and elute the plasmids enriched on the silica membrane of the extraction column.

[0251] The eluted plasmid can then be transformed into competent cells and plated again according to Implementation Method 1 to obtain a monoclonal strain that can express the protein.

[0252] In summary, this application utilizes a functional zoning layout, specifically by setting up a pipetting workstation and peripheral equipment island 1, a plasmid extraction and cell transformation plating workstation 2, a colony picker 3, and a constant temperature and humidity automated incubator 4. This approach separates liquid handling, strain handling, picking, and culture operations into different devices based on functional zoning and throughput levels. Each zone can operate independently or be connected as a whole to run the entire process. The four functional zones are connected in series via a central robotic arm 5, ensuring both process continuity and preventing mutual interference. The DNA molecular-level processing is performed in a traditional pipetting workstation and peripheral equipment island 1, without involving live cells. Processes requiring live colony handling, such as cell lysis for plasmid extraction, heat shock transformation, and culture dish plating, are performed in plasmid extraction and cell transformation plating workstation 2. This workstation effectively integrates plasmid extraction, plasmid transformation into competent cells, and plating of transformed cells. Compared to other manufacturers' solutions that use separate equipment pieced together with island robotic arms 124, this significantly improves processing efficiency by eliminating the need for the robotic arms 124 to move between different devices. Colony picking, while simple, requires a large mechanical travel and is performed separately in the colony picker 3. Cell culture requires a stable culture environment and is performed separately in a constant temperature and humidity automated incubator 4.

[0253] In the above embodiments, the descriptions of each embodiment have different focuses. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0254] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0255] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of this patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A molecular cloning device, characterized by, The application relates to a robot system for automatic extraction and transformation of plasmid and cell, comprising: a pipetting workstation (11), a peripheral device island (12) for storing and transferring consumables, and a mobile station (13) capable of interacting with the central mechanical arm (5) to transfer samples, wherein the peripheral device island (12) comprises an island mechanical arm (124) for transferring reagents and consumables, and the mobile station (13) is provided with a film sealing and tearing device and a centrifuge for centrifugation; a PCR instrument arranged in the pipetting workstation (11) or the mobile station (13); a plasmid extraction and cell transformation coating workstation (2) for extracting plasmid and transforming and coating competent cells; a colony picking instrument (3) for picking colonies in a culture dish into liquid medium of a deep-well plate; a constant-temperature and constant-humidity automatic incubator (4) for automatically culturing competent cells under constant-temperature and constant-humidity conditions; and a central mechanical arm (5) for transferring samples and / or consumables between the mobile station (13), the plasmid extraction and cell transformation coating workstation (2), the colony picking instrument (3) and the constant-temperature and constant-humidity automatic incubator (4). The pipetting workstation (11) comprises a pipetting workbench and the following components connected to the pipetting workbench: a first magnetic rack (1104) for adsorbing magnetic beads; a first metal bath (1105) for constant-temperature preservation of reagents under the condition of 4-60 DEG C; an uncapping module (1106) for uncapping a cryopreservation tube; a first pipetting gun (1107) for pipetting; a first plate transfer clamp jaw (1108) for transferring container consumables or consumable carriers between the mobile stations (13) of the pipetting workstation (11); a PCR product separation and purification device for separating and purifying PCR products; a first heating and oscillation module (1100) for heating a hole plate and rotating and mixing the hole plate; a sample adding needle (1111) connected to a multi-channel rotary valve and used for adding low-value reagents such as buffer; a waste liquid needle (1112) connected to a peristaltic pump and used for removing waste liquid; and a vacuum suction disc (1113) used for opening a culture dish cover. Optionally, the pipetting workbench is further provided with a first guide component capable of transverse movement and longitudinal movement in the horizontal direction, and the uncapping module (1106), the first pipetting gun (1107), the first plate transfer clamp jaw (1108), the sample adding needle (1111) and the waste liquid needle (1112) are arranged on the first guide component respectively. Further optionally, the first guide component comprises a first guide rail extending in the horizontal direction in the transverse direction, a second guide rail slidably connected to the first guide rail and a first sliding seat slidably connected to the second guide rail, and the uncapping module (1106), the first pipetting gun (1107), the first plate transfer clamp jaw (1108), the sample adding needle (1111) and the waste liquid needle (1112) are arranged on the first sliding seat respectively. The pipetting workstation (11) further comprises at least one of the following structures: ​ ​ 2. The molecular cloning device of claim 1, wherein, ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 3. The molecular cloning device of claim 2, wherein, ​ (1) A passive consumable plate base (1101) connected to the pipetting workstation for storing consumables without power supply requirements; (2) A multi-station inclined table (1102) connected to the pipetting workstation for tilting the well plate to concentrate the liquid in the well plate to one side edge; Optionally, the multi-station inclined table is connected with an inclined table driving component, the multi-station inclined table is rotatably connected to the pipetting workstation, and the multi-station inclined table can be rotated to the required angle of inclination and maintained under the driving of the inclined table driving component; (3) A garbage can guide groove (1103) connected to the pipetting workstation for accommodating a garbage can (27) for storing waste consumables; (4) The agar gel electrophoresis apparatus (1109) has double-row comb holes; (5) A sample adding needle (1111) and waste liquid needle (1112) cleaning module (1110) connected to the pipetting workstation for cleaning the sample adding needle (1111) and / or waste liquid needle (1112); (6) The cap opening module (1106) can realize independent Z-axis single-channel cryogenic tube rotary cap opening, and the cap opening module (1106) can move independently in the vertical direction and open or close the cap of one capped cryogenic tube at a time; Optionally, the cap opening module (1106) includes a first cap opening clamp jaw, a second cap opening clamp jaw, a first cap opening driving component, and a second cap opening driving component, the first cap opening driving component and the second cap opening driving component are respectively installed on the pipetting workstation, the first cap opening clamp jaw is connected with the first cap opening driving component, the first cap opening driving component is used to drive the first cap opening clamp jaw to move and clamp the test tube, the second cap opening driving component is connected with the second cap opening clamp jaw to drive the second cap opening clamp jaw to move and clamp the test tube cap, and the second cap opening driving component drives the second cap opening to move reversely to open or close the test tube cap; (7) The first pipetting gun (1107) includes a first multi-channel variable-distance independent Z-axis pipetting gun, and the first pipetting gun (1107) can control each pipetting head to move independently in the vertical direction and can adjust the distance between different channels as needed to adapt to different specifications of container consumables; (8) The sample adding needle (1111) includes an independent Z-axis sample adding needle (1111), and the sample adding needle (1111) can move independently in the vertical direction; (9) The waste liquid needle (1112) includes an independent Z-axis waste liquid needle (1112), and the sample adding needle (1111) can move independently in the vertical direction.

4. The molecular cloning device of claim 1, wherein, The peripheral device island table (12) satisfies at least one of the following conditions: (1) The peripheral device island table (12) includes: A capped test tube cap opening machine (121) for opening the cap of a test tube; A large-capacity light-shielded high-low temperature box (122) for incubating samples in a well plate and temporarily storing low-temperature reagents; A consumable tower (123) for storing consumables including a gun head box and a well plate; And, a transfer station (125) for interacting with the central robot (5) to sample, the island robot (124) can get reagent sample containers or reagent consumable containers at the cap-sealing test tube cap-sealing machine (121), the large-capacity light-shielded high-temperature box (122) and transfer to the transfer station (125); (2) The peripheral device island (12) is arranged downstream of the pipetting workstation (11); (3) The peripheral device island (12) further comprises an island workbench, and the island robot (124) is slidably connected to the island robot (124) to transfer reagents and consumables between the large-capacity light-shielded high-temperature box (122) and the consumable tower (123).

5. The molecular cloning device of claim 1, wherein, The moving station (13) comprises a plurality of, and one of the PCR instrument, the film sealing and tearing device and the centrifuge is respectively arranged at different moving stations (13).

6. The molecular cloning device of claim 1, wherein, The plasmid extraction and cell transformation coating workstation (2) comprises an extraction and transformation coating workbench and the following devices connected to the extraction and transformation coating workbench: A second pipette (21) for pipetting; A second transfer plate clamping jaw (22) for clamping and moving a transfer jig containing a competent cell tube; A second heating and shaking module (23) for heating and shaking a multi-well plate; A second magnetic stand (24) for adsorbing magnetic beads; A plasmid extraction device (26) for automatically extracting plasmids; A cell transformation mechanism (28) for transforming competent cells; And a coating module (29) for coating competent cells.

7. The molecular cloning device of claim 6, wherein, Among them, The plasmid extraction device (26) comprises: A filter column (261) for filtering cell lysate; A collection tube (262) located below the filter column (261) to collect plasmids filtered by the filter column (261) and purify plasmids by magnetic beads; A DNA extraction column (263) for enriching plasmids from the collection tube (262) on a silica gel membrane after positive pressure; A waste tank (264) for collecting waste liquid from the filter column (261); And a plasmid collection plate (265) for collecting plasmids purified by magnetic beads from the collection tube (262) or collecting plasmids on the silica gel membrane from the DNA extraction column (263).

8. The molecular cloning device of claim 7, wherein, The cell transformation mechanism (28) comprises a second metal bath, which is used for cold or hot bathing of the competent cell tube clamped on the transfer jig; Optionally, the second metal bath comprises a cold metal bath mechanism (281) and a hot metal bath mechanism (282); the cold metal bath mechanism (281) is used for cold bathing of the competent cell tube clamped on the transfer jig, and the hot metal bath mechanism (282) is used for hot bathing of the competent cell tube clamped on the transfer jig; Optionally, the cold metal bath mechanism (281) has a plurality of channel stations, so that the cold metal bath mechanism (281) can match the 96-well throughput of the plasmid collection plate (265).

9. The molecular cloning device of any one of claims 6-8, wherein, The cell transformation and coating workstation (2) further satisfies at least one of the following conditions: (1) The coating plate module (29) comprises a lifting-rotating module coating head, which is installed on a protractor, and the protractor can rotate to move the lifting-rotating module coating head to a cleaning station, a high-temperature infrared sterilization station, and a drying station, and respectively perform cleaning, high-temperature infrared sterilization, and drying of the lifting-rotating module coating head; (2) The second pipette (21) comprises a second multi-channel variable-distance independent Z-axis pipette, which can control each pipetting head to independently move in the vertical direction and can adjust the distance between different channels as needed to adapt to different specifications of multi-well plates; (3) The extraction and conversion coating plate workstation is further provided with a second guide component capable of moving horizontally in the horizontal direction and vertically, and the second pipette (21) and the second plate clamping jaw (22) are respectively arranged on the second guide component; (4) The second guide component comprises a third guide rail extending horizontally in the horizontal direction, a fourth guide rail slidably connected to the third guide rail, and a second sliding seat slidably connected to the fourth guide rail, and the second pipette (21) and the second plate clamping jaw (22) are respectively arranged on the second sliding seat.

10. The molecular cloning device of any one of claims 6-8, wherein, The plasmid extraction and cell conversion coating plate workstation (2) further comprises at least one of the following structures: (1) A pipette tip box support (25) for storing a pipette tip box; (2) A garbage can (27) for storing waste consumables and waste filter columns.

11. The molecular cloning device of any one of claims 1 to 8, wherein, The colony picking instrument (3) has: A petri dish inlet and outlet (311) for interacting with the central mechanical arm (5) to pick a colony; A culture solution inlet and outlet (312) for interacting with the central mechanical arm (5) to store a culture solution container for storing the picked colony.

12. The molecular cloning device of any one of claims 1 to 8, wherein, The colony picking instrument (3) comprises: A colony picking workstation (320) for carrying a plurality of petri dishes; A feeding mechanism (310) arranged on the colony picking workstation (320), the feeding mechanism (310) is used for continuously outputting materials (361); A bacteria picking mechanism (330) arranged above the colony picking workstation (320), the bacteria picking mechanism (330) is used for receiving the materials (361) output by the feeding mechanism (310), and driving the head end of the materials (361) to output a preset length towards the colony picking workstation (320) to form a bacteria picking piece (362) for picking bacteria; A driving mechanism (350) connected with the bacteria picking mechanism (330), the driving mechanism (350) is used for driving the bacteria picking mechanism (330) to move, so that the bacteria picking mechanism (330) drives the bacteria picking piece (362) to pick a colony from one of the petri dishes, and / or, the bacteria picking mechanism (330) drives the bacteria picking piece (362) to plant the colony in another petri dish; And a cutting mechanism (340) for separating the bacteria picking member (362) from the material (361).

13. The molecular cloning device of claim 12, wherein, The feeding mechanism (310) comprises: A conduit (312) having one end connected to the bacteria picking mechanism (330); An extruding mechanism (311) disposed on the bacteria colony picking workbench (320) and connected to the other end of the conduit (312), the extruding mechanism (311) being used for conveying the material (361) along the conduit (312) to the bacteria picking mechanism (330).

14. The molecular cloning device of claim 13, wherein, The extruding mechanism (311) comprises a driving wheel (3113) rotatably disposed on the bacteria colony picking workbench (320) and a driven wheel (3114) disposed at intervals from the driving wheel (3113), a conveying channel for conveying the material (361) being formed between the driving wheel (3113) and the driven wheel (3114), the driving wheel (3113) being able to be driven to rotate, and the driving wheel (3113) being able to drive the material (361) to advance in cooperation with the driven wheel (3114) when the driving wheel (3113) is driven.

15. The molecular cloning apparatus of claim 13, wherein, The cutting mechanism (340) comprises a cutting knife (341) movably disposed on the bacteria colony picking workbench (320), the cutting knife (341) being able to act to cut the material (361) at the end of the conduit (312).

16. A molecular cloning method, characterized by, The molecular cloning device comprises the following steps: The island table mechanical arm (124) is controlled to transfer the multi-well plate from the subzero temperature environment of the peripheral equipment island table (12) to a centrifuge, the centrifuge is controlled to centrifuge, the island table mechanical arm (124) is controlled to transfer the multi-well plate to a film sealing and tearing device, the film sealing and tearing device is controlled to remove the film on the multi-well plate, the island table mechanical arm (124) is controlled to transfer the multi-well plate in the film sealing and tearing device to a first metal bath (1105) in the pipetting workstation (11) with a first preset temperature, the island table mechanical arm (124) is controlled to transfer the low-temperature reagent in the peripheral equipment island table (12) to the first metal bath (1105), the pipetting workstation (11) is controlled to transfer the required low-temperature reagent into the wells of the multi-well plate, and other required reagents are added to prepare a PCR reaction system, the island table mechanical arm (124) is controlled to send the multi-well plate into a PCR instrument, the PCR instrument is controlled to amplify, and the PCR amplification product is separated and purified by the agarose gel electrophoresis instrument (1109) of the pipetting workstation (11), and the purified fragment is transferred to a new PCR well plate; controlling the island mechanical arm (124) to transfer the PCR hole plate containing the purified fragments to the first metal bath (1105) with the temperature set to the first preset temperature, and controlling the island mechanical arm (124) to transfer other 0°C below stored enzyme cutting system related reagents from the peripheral equipment island (12) to the first metal bath (1105); controlling the pipetting workstation (11) to transfer the enzyme cutting system related reagents to the PCR hole plate to prepare the enzyme cutting system, and reacting in the first metal bath (1105) with the temperature set to the second preset temperature for 2h~3h, recovering the enzyme cutting product; constructing a ligation reaction system containing the enzyme cutting product and mixing in a PCR tube, and reacting at room temperature overnight for 8h~12h to obtain a plasmid integrating the target DNA fragment into the vector plasmid, and extracting the plasmid; controlling the central mechanical arm (5) to send the new multi-hole plate containing the plasmid into the plasmid extraction and cell transformation coating workstation (2) for transformation, and transferring the transformed competent cells to the culture dish of the coating module (29); controlling the coating module (29) to uniformly coat the transformed competent cells, and performing cleaning and sterilization procedures; controlling the central mechanical arm (5) and the island mechanical arm (124) to cooperate to invert the coated culture dish and transfer it to the constant temperature and humidity automatic incubator (4) for culture; After the culture is completed, controlling the central mechanical arm (5) to sequentially send the culture dish into the colony picking instrument (3) for colony picking, and storing the picked colonies in a deep hole plate containing culture solution.

17. The molecular cloning method of claim 16, wherein, It also satisfies one or several of the following conditions: (1) Before controlling the island mechanical arm (124) to transfer the multi-hole plate from the zero temperature environment of the peripheral equipment island (12) to the centrifuge, the following preparation work is further included: placing the multi-hole plate containing the DNA template and the film and the low-temperature reagent into the large-capacity light-shielded high-low temperature box (122), placing the normal-temperature reagents and consumables into the designated positions of the passive consumable plate site base (1101), and respectively configuring the PCR instrument, the film sealing and tearing device, and the centrifuge at the moving station (13); (2) When controlling the island mechanical arm (124) to transfer the multi-hole plate from the zero temperature environment of the peripheral equipment island (12) to the centrifuge of the moving station (13), the following steps are specifically included: controlling the island mechanical arm (124) to transfer the multi-hole plate from the zero temperature environment in the large-capacity light-shielded high-low temperature box (122) of the peripheral equipment island (12) to the centrifuge of the moving station (13); Optionally, controlling the island mechanical arm (124) to clamp a special hole plate for balancing from the consumable tower (123) for balancing the centrifuge; (3) When controlling the pipetting workstation (11) to transfer the required low-temperature reagents into the holes of the multi-hole plate, the following steps are included: controlling the cap opening module (1106) to unscrew the cap of the low-temperature reagent, controlling the first pipetting gun (1107) to suck the required low-temperature reagents into the holes of the multi-hole plate, and controlling the cap opening module (1106) to reseal the cap; (4) when adding other required reagents to the wells of the multi-well plate to prepare a PCR reaction system, comprising the following steps: controlling the first pipettor (1107) to add other required reagents to prepare a PCR reaction system; (5) when separating and purifying the PCR amplification product by the agarose gel electrophoresis instrument (1109) of the pipetting workstation (11) and transferring the purified fragment to a new PCR well plate, comprising the following steps: controlling the island platform mechanical arm (124) to transfer the multi-well plate after amplification to the pipetting workstation (11), and controlling the first pipettor (1107) to transfer the amplification product in the multi-well plate to the first row of comb wells of the agarose gel electrophoresis instrument (1109); controlling the agarose gel electrophoresis instrument (1109) to perform electrophoresis, separating the target molecular weight band under the monitoring of the imaging module until the target molecular weight band reaches the second row of comb wells of the agarose gel electrophoresis instrument (1109), and then ending the electrophoresis; controlling the first pipettor (1107) to transfer the purified fragment in the second row of comb wells to a new PCR well plate; (6) when controlling the pipetting workstation (11) to transfer the reagents related to the enzyme digestion system to the PCR well plate to prepare the enzyme digestion system, comprising the following steps: controlling the island platform mechanical arm (124) to transfer the PCR well plate containing the purified fragment to the first metal bath (1105) with a first preset temperature, and controlling the island platform mechanical arm (124) to transfer other enzyme digestion system related reagents stored below 0°C from the large-capacity light-shielded high-low temperature box (122) to the first metal bath (1105); controlling the cap opening module (1106) to unscrew the cap corresponding to the enzyme digestion system related reagent, controlling the first pipettor (1107) to suck the enzyme digestion system related reagent into the PCR well plate, and controlling the cap opening module (1106) to screw the cap of the enzyme digestion system related reagent back; controlling the first pipettor (1107) to continue adding other required reagents to the PCR well plate to prepare the enzyme digestion system; and controlling the PCR well plate to react in the first metal bath (1105) with a second preset temperature for 2h-3h; (7) when recovering the enzyme digestion product, comprising the following steps: recovering the enzyme digestion product by using the agarose gel recovery method; (8) when constructing a ligation reaction system containing the enzyme digestion product, comprising the following steps: operating on ice, controlling the island platform mechanical arm (124) and the first pipettor (1107) to mix the ligation reaction system in a new PCR tube, and controlling the island platform mechanical arm (124) to move the PCR tube to the first metal bath (1105) with a room temperature setting or to react at room temperature overnight for 8h-12h; (9) when controlling the central mechanical arm (5) to send a new multi-well plate containing a plasmid into the plasmid extraction and cell transformation coating workstation (2) for transformation, comprising the following steps: The competent cell container is loaded onto the tray of the cold metal bath mechanism (281) heat conduction jig, the second pipette (21) is controlled to sequentially suck the plasmid from the delivered multi-well plate and add it into the competent cell container, the second transfer plate clamping jaw (22) is controlled to transfer the cold metal bath mechanism (281) heat conduction jig and the tray to the preheated hot metal bath mechanism (282), and heat shock transformation is performed for 45s~90s; during the transformation process, the constant temperature and humidity automatic incubator (4) is controlled to pop out the culture dish that has been preloaded into the incubator through the transfer window (41), the central mechanical arm (5) is controlled to deliver the culture dish into the plasmid extraction and cell transformation coating plate workstation (2), the second transfer plate clamping jaw (22) is controlled to place the culture dish on the tray of the coating plate module (29) and open the cover, and the second transfer plate clamping jaw (22) is controlled to transfer the heat conduction jig and the tray in the hot metal bath mechanism (282) after the heat shock transformation is completed to the cold metal bath mechanism (281) for cooling for 2min~10min; (10) When the transformed competent cells are transferred to the culture dish of the coating plate module (29), the following steps are specifically included: the second pipette (21) is controlled to transfer the transformed competent cells to the culture dish of the coating plate module (29); (11) When the plasmid extraction and cell transformation coating plate workstation (2) is transformed, the pipetting workstation and the peripheral equipment island (12) perform the operation of the next batch of samples; (12) When the cleaning and sterilization process is performed, the following steps are included: during cleaning, the spray tank maintains negative pressure, and an alkaline cleaning agent and pure water are used for spray washing; during sterilization, an infrared sterilization lamp is used for sterilization at 450℃~480℃.

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