阅读说明：本技术 一种dna提纯方法以及专用设备 (DNA purification method and special equipment ) 是由 杨冰 杨博然 闫亮亮 李文佳 于 2019-08-20 设计创作，主要内容包括：本发明涉及一种DNA提纯方法,包括以下步骤：⑴通电进行第一次电泳；⑵将所需的含有DNA分子片段的凝胶进行切块；⑶切块进行第二次电泳,使DNA分子片段移动至缓冲液中；⑷对步骤⑶的缓冲液进行吸附柱吸附或磁珠吸附；⑸清洗后下次试验使用。本发明中,将第一次电泳的凝胶切块取出,并在缓冲液中进行第二次电泳,使DNA分子片段在电场的作用下移动缓冲液中,再由吸附柱或磁珠进行DNA分子片段的分离、提纯,收集的DNA分子片段纯度比传统的方法提高了5～10％,分离、提纯的时间比传统的方法节省了50％以上。(the invention relates to a DNA purification method, which comprises the following steps: performing first electrophoresis on power; dicing the gel containing the DNA molecule fragments; performing secondary electrophoresis on the cut blocks to move the DNA molecular fragments into a buffer solution; fourthly, performing adsorption column adsorption or magnetic bead adsorption on the buffer solution obtained in the step three; and fifthly, carrying out next test after washing. In the invention, the gel blocks obtained by the first electrophoresis are taken out and subjected to the second electrophoresis in the buffer solution, so that the DNA molecular fragments move in the buffer solution under the action of an electric field, and then the separation and purification of the DNA molecular fragments are carried out by the adsorption column or the magnetic beads, the purity of the collected DNA molecular fragments is improved by 5-10% compared with that of the traditional method, and the separation and purification time is saved by more than 50% compared with that of the traditional method.)

1. A DNA purification method is characterized in that: the method comprises the following steps:

adding a DNA sample into each agarose gel, and electrifying to perform primary electrophoresis;

Secondly, cutting the required gel containing the DNA molecular fragments by electrophoresis for the first time;

Thirdly, carrying out secondary electrophoresis on the cut blocks taken from each agarose gel to move the DNA molecular fragments into the buffer solution;

Fourthly, performing adsorption column adsorption or magnetic bead adsorption on the buffer solution obtained in the step three;

and fifthly, carrying out next test after washing.

2. The method for purifying DNA according to claim 1, wherein: the buffer solution during the first electrophoresis comprises 0.04-0.09M of TRIS-HCL, 0001-0.005M of EDTA, 0.15M of glycine, 0.05M of boric acid and 0.2M of dithiothreitol;

the buffer solution for the second electrophoresis comprises 0.04-0.09M TRIS-HCL, 0001-0.005M EDTA, 0.15M glycine, 0.05M boric acid, 0.2M dithiothreitol and 0.001M sodium thiosulfate;

the second electrophoresis uses intermittent pulse voltages.

3. a dedicated apparatus for use in the DNA purification method according to claim 1 or 2, characterized in that: the structure of the electrophoresis equipment during the first electrophoresis is as follows: including end box, electrophoresis box and upper cover, a plurality of electrophoresis boxes are placed to the spaced in the end box, and the inside plate electrode that sets up in every electrophoresis box both sides is connected with the binding post electricity that sets up with the end box lateral wall of homonymy respectively, and this binding post is connected with the power electricity, lock upper cover on the end box.

4. The apparatus for DNA purification according to claim 3, wherein: the upper cover is provided with a plurality of temperature sensors which are aligned with the electrophoresis boxes, the lower end of each temperature sensor extends below the liquid level of the buffer solution at the position of the gel tray, and the output ends of the plurality of temperature sensors are connected with the sockets arranged on the upper cover.

5. A dedicated apparatus for use in the DNA purification method according to claim 1 or 2, characterized in that: the structure of the electrophoresis device in the second electrophoresis is as follows: including cooling box, electrophoresis box and upper cover, place at least one electrophoresis box in the cooling box, set up a plurality of mutual isolation's electrophoresis cell in the electrophoresis box, the plate electrode that every electrophoresis cell both sides set up respectively with the binding post of the cooling box lateral wall setting of homonymy realize the electricity through the mode of crimping or gliding mode to be connected, this binding post is connected with the power electricity, the lock upper cover on the cooling box, remain the clearance between electrophoresis box and the cooling box, this clearance respectively with the coolant import and the coolant export intercommunication that cooling box surface set up.

6. the apparatus for DNA purification method according to claim 5, wherein: when the electrical connection is in a crimping mode: the inner parts of the side walls at the two sides of the electrophoresis box are respectively provided with a bending plate, one side of the bending plate in the electrophoresis box is provided with end parts which are bent downwards and extend into the electrophoresis tank and form electrode plates, and the end parts at the other side of the bending plate are electrically connected with the wiring terminals;

The outer edge of the wiring terminal is provided with external threads and is connected to the surface of the side wall where the cooling box and the wiring board are aligned in a meshed mode, the end portion of the inner side of the wiring terminal is in contact with the wiring board, the end portion of the outer side of the wiring terminal is provided with a wire inserting hole and a fixing bolt, the wire inserting hole is used for inserting the end portion of a cable connected with a power supply, and the fixing bolt is used for fixing the end portion of the cable in the end portion.

7. the apparatus for DNA purification method according to claim 5, wherein: when electrically connected in a sliding manner: the other side end of the bending plate is provided with a connecting contact point, and the outer side surface of the connecting contact point is arc-shaped;

the outer edge of the wiring terminal is provided with external threads and is meshed with the surface of the side wall where the cooling box is aligned to the arc, the end part of the inner side of the wiring terminal is in sliding contact with the arc, the end part of the outer side of the wiring terminal is provided with a wire inserting hole and a fixing bolt, the wire inserting hole is used for inserting the end part of a cable connected with a power supply, and the fixing bolt is used for fixing the end part of the cable in the end part of the outer side of the wiring terminal.

8. The apparatus for DNA purification method according to claim 6 or 7, wherein: the electrophoresis box is characterized in that at least one sealing ring is arranged at the upper end of the outer edge of the electrophoresis box from top to bottom, the outer edge of the electrophoresis box below the sealing ring is provided with a recess, the inner edge of the cooling box aligned with the recess is provided with a recess, the gap comprises two recesses, the bottom surface of the electrophoresis box and the inner bottom surface of the cooling box, the lower end of the surface of one side of the cooling box is provided with a refrigerant inlet, and the upper end of the surface of the other side of the cooling.

9. a dedicated apparatus for use in the DNA purification method according to claim 1 or 2, characterized in that: the step fifthly cleaning structure is as follows: the DNA electrophoresis cleaning device comprises a cleaning box, an end cover and a transverse motion module, wherein the end cover is buckled at an opening at the upper end of the cleaning box, the transverse motion module is transversely arranged on the upper end surface of the end cover, the lower end of a liquid outlet pipe arranged on the transverse motion module extends into the cleaning box, a plurality of micro spray holes are arranged at the lower end of the liquid outlet pipe, the liquid outlet pipe is communicated with a liquid outlet of a liquid pump, a liquid inlet of the pump is communicated with a cleaning liquid source, and a plurality of DNA electrophoresis tanks are transversely arranged at the bottom in the cleaning box;

Soaking the DNA electrophoresis cell by using cleaning liquid placed in the cleaning box; the transverse motion module drives the liquid outlet pipe to move transversely, and cleaning liquid sprayed by the micro-spray holes is used for carrying out spray-washing treatment on the DNA electrophoresis cell.

10. The apparatus for purifying DNA according to claim 9, wherein: the transverse motion module is a linear motor, a transverse boss is arranged on the upper end face of the end cover, a bottom plate of the linear motor is mounted on the boss, a guide rail is arranged on the bottom plate, a motion plate is arranged on a slide block which is arranged on the guide rail in a sliding mode, a liquid pump is arranged on the motion plate, and a liquid outlet pipe communicated with a liquid outlet of the liquid pump extends into the cleaning box from a transverse long hole arranged on the boss and a transverse long hole arranged in an aligned mode of the end cover;

A longitudinal arc-shaped pipe is arranged at the lower end of the liquid outlet pipe, the arc-shaped surface of the arc-shaped pipe faces the bottom of the cleaning box, and a plurality of micro spray holes are formed in the arc-shaped surface;

a gap is reserved between the bottom surface of the electrophoresis cell and the bottom surface in the cleaning box, and a liquid outlet is arranged on the side wall of the cleaning box at any side of the gap;

the bottom of the cleaning box is provided with a heating rod, and the cleaning box is internally provided with a liquid level probe and a temperature sensor.

Technical Field

The invention belongs to the technical field of electrophoresis, and relates to a DNA electrophoretic separation method, in particular to a DNA purification method and special equipment.

background

DNA gel electrophoresis is commonly used for analytical purposes, and can also be used as a preparation technique, and the structure of the DNA gel electrophoresis is as follows: set up an electrode plate respectively in the both sides of electrophoresis cell, the middle part of electrophoresis cell sets up a arch, places the gel tray on the arch, places agarose gel in the gel tray, places the DNA sample in the groove of agarose gel, puts into the buffer solution of sufficient volume in the electrophoresis tank, makes its gel surface that submerges the gel tray. In the test, the electrode plates are electrified, the DNA molecules on the gel move towards the positive direction by the electric field between the two electrode plates, and the different DNA molecule fragments are separated out due to different molecules and configurations of the different DNA molecule fragments and different moving speeds in the electric field.

the both sides of current electrophoresis cell set up a contact pin respectively, and the plate electrode of homonymy is connected to every contact pin lower extreme, and in the upper end of every contact pin imbeds the socket that the upper cover counterpointed the setting behind electrophoresis cell lock upper cover, two sockets of upper cover both sides were connected the anodal of power and the binding post of negative pole respectively. The pins are inserted into the holes of the socket, so it is not convenient to fasten the pins.

In addition, in the conventional DNA purification process, electrophoresis is performed first to locate DNA molecule fragments at different positions of a gel, then the gel where the DNA molecule fragments are located is cut into pieces, the pieces are taken out and heated to be dissolved in a solvent, and the solvent is adsorbed by an adsorption column or magnetic beads to extract desired DNA molecule fragments. The adsorption column is used for adsorbing DNA molecular fragments by using the DNA adsorption column, then the DNA molecular fragments are recovered after elution, and the magnetic bead adsorption is used for adsorbing the DNA molecular fragments on the surface of magnetic beads, and then the DNA molecular fragments are recovered after elution. In any of the above separation methods, agarose is dissolved in a solvent after heating, and becomes an impurity of a DNA molecule fragment during adsorption, which is not favorable for a later test.

disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a DNA purification method which can reduce agarose impurities and can quickly recover DNA molecular fragments by carrying out secondary electrophoresis on gel blocks so as to move the DNA molecular fragments into a buffer solution.

the technical scheme adopted by the invention is as follows:

a DNA purification method is characterized in that: the method comprises the following steps:

Adding a DNA sample into each agarose gel, and electrifying to perform primary electrophoresis;

Secondly, cutting the required gel containing the DNA molecular fragments by electrophoresis for the first time;

Thirdly, carrying out secondary electrophoresis on the cut blocks taken from each agarose gel to move the DNA molecular fragments into the buffer solution;

fourthly, performing adsorption column adsorption or magnetic bead adsorption on the buffer solution obtained in the step three;

And fifthly, carrying out next test after washing.

The buffer solution for the first electrophoresis comprises 0.04-0.09M TRIS-HCL, 0001-0.005M EDTA, 0.15M glycine, 0.05M boric acid and 0.2M dithiothreitol;

The buffer solution for the second electrophoresis comprises 0.04-0.09M TRIS-HCL, 0001-0.005M EDTA, 0.15M glycine, 0.05M boric acid, 0.2M dithiothreitol and 0.001M sodium thiosulfate;

The second electrophoresis uses intermittent pulse voltages.

Another object of the present invention is to provide an electrophoresis apparatus for first electrophoresis, which has a structure in which: including end box, electrophoresis box and upper cover, a plurality of electrophoresis boxes are placed to the spaced in the end box, and the inside plate electrode that sets up in every electrophoresis box both sides is connected with the binding post electricity that sets up with the end box lateral wall of homonymy respectively, and this binding post is connected with the power electricity, lock upper cover on the end box.

Furthermore, a plurality of temperature sensors are arranged on the upper cover in alignment with each electrophoresis box, the lower end of each temperature sensor extends below the liquid level of the buffer solution at the position of the gel tray, and the output ends of the plurality of temperature sensors are connected with a socket arranged on the upper cover.

another object of the present invention is to provide an electrophoresis apparatus for second electrophoresis, which has a structure of: including cooling box, electrophoresis box and upper cover, place at least one electrophoresis box in the cooling box, set up a plurality of mutual isolation's electrophoresis cell in the electrophoresis box, the plate electrode that every electrophoresis cell both sides set up respectively with the binding post of the cooling box lateral wall setting of homonymy realize the electricity through the mode of crimping or gliding mode to be connected, this binding post is connected with the power electricity, the lock upper cover on the cooling box, remain the clearance between electrophoresis box and the cooling box, this clearance respectively with the coolant import and the coolant export intercommunication that cooling box surface set up.

Further, in the case of electrical connection by pressure bonding: the inner parts of the side walls at the two sides of the electrophoresis box are respectively provided with a bending plate, one side of the bending plate in the electrophoresis box is provided with end parts which are bent downwards and extend into the electrophoresis tank and form electrode plates, and the end parts at the other side of the bending plate are electrically connected with the wiring terminals;

The outer edge of the wiring terminal is provided with external threads and is connected to the surface of the side wall where the cooling box and the wiring board are aligned in a meshed mode, the end portion of the inner side of the wiring terminal is in contact with the wiring board, the end portion of the outer side of the wiring terminal is provided with a wire inserting hole and a fixing bolt, the wire inserting hole is used for inserting the end portion of a cable connected with a power supply, and the fixing bolt is used for fixing the end portion of the cable in the end portion.

Further, when the electrical connection is a sliding type: the other side end of the bending plate is provided with a connecting contact point, and the outer side surface of the connecting contact point is arc-shaped;

The outer edge of the wiring terminal is provided with external threads and is meshed with the surface of the side wall where the cooling box is aligned to the arc, the end part of the inner side of the wiring terminal is in sliding contact with the arc, the end part of the outer side of the wiring terminal is provided with a wire inserting hole and a fixing bolt, the wire inserting hole is used for inserting the end part of a cable connected with a power supply, and the fixing bolt is used for fixing the end part of the cable in the end part of the outer side of the wiring terminal.

And the upper end of the outer edge of the electrophoresis box is provided with at least one sealing ring from top to bottom, the outer edge of the electrophoresis box below the sealing ring is provided with a recess, the inner edge of the cooling box aligned with the recess is provided with a recess, the gap comprises two recesses, the bottom surface of the electrophoresis box and the inner bottom surface of the cooling box, the lower end of the surface of one side of the cooling box is provided with a refrigerant inlet, and the upper end of the surface of the other side of the cooling box is provided with a refrigerant outlet.

another object of the present invention is to provide a special apparatus for cleaning, which has the structure that: the DNA electrophoresis cleaning device comprises a cleaning box, an upper cover and a transverse motion module, wherein the upper cover is buckled at an opening at the upper end of the cleaning box, the transverse motion module is transversely arranged on the upper end surface of the upper cover, the lower end of a liquid outlet pipe arranged on the transverse motion module extends into the cleaning box, a plurality of micro spray holes are arranged at the lower end of the liquid outlet pipe, the liquid outlet pipe is communicated with a liquid outlet of a liquid pump, a liquid inlet of the liquid pump is communicated with a cleaning liquid source, and a plurality of DNA electrophoresis tanks are transversely arranged at the bottom in the cleaning box;

Soaking the DNA electrophoresis cell by using cleaning liquid placed in the cleaning box; the transverse motion module drives the liquid outlet pipe to move transversely, and cleaning liquid sprayed by the micro-spray holes is used for carrying out spray-washing treatment on the DNA electrophoresis cell.

The transverse motion module is a linear motor, a transverse boss is arranged on the upper end face of the upper cover, a bottom plate of the linear motor is mounted on the boss, a guide rail is arranged on the bottom plate, a motion plate is arranged on a slide block which is arranged on the guide rail in a sliding mode, a liquid pump is arranged on the motion plate, and a liquid outlet pipe communicated with a liquid outlet of the liquid pump extends into the cleaning box from a transverse long hole formed in the boss and a transverse long hole formed in the upper cover in an opposite mode;

a longitudinal arc-shaped pipe is arranged at the lower end of the liquid outlet pipe, the arc-shaped surface of the arc-shaped pipe faces the bottom of the cleaning box, and a plurality of micro spray holes are formed in the arc-shaped surface;

a gap is reserved between the bottom surface of the electrophoresis cell and the bottom surface in the cleaning box, and a liquid outlet is arranged on the side wall of the cleaning box at any side of the gap;

the bottom of the cleaning box is provided with a heating rod, and the cleaning box is internally provided with a liquid level probe and a temperature sensor.

The invention has the advantages and positive effects that:

1. The electrophoresis equipment for the first electrophoresis in the method comprises a bottom box, wherein a plurality of electrophoresis boxes can be placed in the bottom box, the lower end of a bending plate at the outer edge of each electrophoresis box is electrically connected with a wiring terminal, an upper cover is only used for being buckled on the bottom box, the lower end edge of the upper cover shields the wiring terminals, a plurality of electrophoresis boxes in the bottom box can be used for separating DNA molecular fragments in a large amount at one time, the upper cover is convenient to buckle, in addition, a temperature sensor is arranged in a gel tray area, the temperature measurement of the area is carried out through the temperature sensor, and impurities generated by the melting of agarose gel due to the overhigh temperature of a buffer solution at the area are avoided.

2. The electrophoresis equipment for the second electrophoresis in the method is characterized in that a plurality of electrophoresis boxes are arranged in a cooling box, the outer side of each electrophoresis box is electrically connected with a connecting terminal on the cooling box in a crimping or sliding mode, a plurality of electrophoresis cells are separated from each electrophoresis box by partition plates, the volumes of the electrophoresis cells can be the same or different, electrode plates are arranged on two sides of each electrophoresis cell, the electrified voltage of the electrode plates can be properly increased so as to improve the moving speed of DNA molecular fragments, the introduction of a refrigerant avoids the problem that gel is dissolved in buffer solution due to the rising of the temperature of the buffer solution, and the recovery purity of the DNA molecular fragments in the later period is ensured.

3. In the cleaning equipment in the method, the liquid pump is used for sucking various liquids, the temperature sensor is used for detecting the temperature of the cleaning liquid, the heating rod is used for heating the cleaning liquid, the valve is used for discharging the cleaning liquid, all electric parts are automatically controlled by the control module, and due to the fact that the water flow coverage area of the micro-spraying holes is large and the flow speed is high, the cleaning equipment has a good cleaning effect on the spaces in the electrophoresis tanks with different volumes in each electrophoresis box.

4. In the method, the gel blocks obtained by the first electrophoresis are taken out and subjected to the second electrophoresis in the buffer solution, so that the DNA molecular fragments move in the buffer solution under the action of an electric field, and then the separation and purification of the DNA molecular fragments are carried out by an adsorption column or magnetic beads, the purity of the collected DNA molecular fragments is improved by 5-10% compared with that of the traditional method, and the separation and purification time is saved by more than 50% compared with that of the traditional method.

Drawings

FIG. 1 is a schematic structural view of the present invention (with the upper cover removed);

FIG. 2 is a bottom view of FIG. 1;

FIG. 3 is a schematic view of the structure of FIG. 1 with all of the electrophoresis cells removed;

FIG. 4 is a left side view of FIG. 2;

FIG. 5 is an enlarged cross-sectional view of FIG. 4;

FIG. 6 is an enlarged view of section I of FIG. 5;

FIG. 7 is a schematic view of the structure of an electrode plate;

FIG. 8 is a schematic view of the structure of FIG. 2 after it has been snapped onto the lid;

FIG. 9 is a state diagram of two electrophoresis cells;

FIG. 10 is a schematic view showing the structure of an electrophoresis apparatus for the second electrophoresis (in a manner of crimping, with the upper cover removed);

FIG. 11 is a schematic diagram of the structure of FIG. 10 with only one electrophoresis cassette;

FIG. 12 is a left side view of FIG. 11;

FIG. 13 is a rear view of FIG. 12;

FIG. 14 is an enlarged cross-sectional view of FIG. 12;

FIG. 15 is a view showing a positional relationship between the separators and the electrode plates of FIG. 14;

FIG. 16 is a schematic view showing the structure of an electrophoresis apparatus for the second electrophoresis (slide mode, with the upper cover removed);

FIG. 17 is a schematic diagram of the structure of FIG. 16 with only one electrophoresis cassette;

FIG. 18 is a left side view of FIG. 17;

FIG. 19 is a rear view of FIG. 18;

FIG. 20 is an enlarged cross-sectional view of FIG. 18;

FIG. 21 is a view showing a positional relationship between the separators and the electrode plates of FIG. 20;

FIG. 22 is a schematic view of the structure of the cleaning apparatus;

FIG. 23 is a left side view of FIG. 22;

FIG. 24 is a cross-sectional view of FIG. 22;

FIG. 25 is a cross-sectional view of FIG. 23;

Fig. 26 is a control schematic of fig. 24.

Detailed Description

the present invention is further illustrated by the following examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.

A DNA purification method is disclosed, as shown in FIGS. 1-26, and the innovation of the invention lies in: the method comprises the following steps:

adding a DNA sample into each agarose gel, and electrifying to perform primary electrophoresis;

secondly, cutting the required gel containing the DNA molecular fragments by electrophoresis for the first time;

thirdly, carrying out secondary electrophoresis on the cut blocks taken from each agarose gel to move the DNA molecular fragments into the buffer solution;

Fourthly, performing adsorption column adsorption or magnetic bead adsorption on the buffer solution obtained in the step three;

and fifthly, carrying out next test after washing.

wherein the buffer solution during the first electrophoresis comprises 0.04-0.09M TRIS-HCL, 0001-0.005M EDTA, 0.15M glycine, 0.05M boric acid and 0.2M dithiothreitol. The buffer solution for the second electrophoresis comprises 0.04-0.09M TRIS-HCL, 0001-0.005M EDTA, 0.15M glycine, 0.05M boric acid, 0.2M dithiothreitol and 0.001M sodium thiosulfate. The second electrophoresis uses intermittent pulsed voltages.

the two buffers are specially configured, and are very stable by matching of the components, so that the electrophoresis can be carried out for a long time. And a proper pH environment is provided for electrophoresis, so that the mobility of the DNA molecular fragments is improved by 3-5% compared with the traditional formula, and the required DNA molecular fragments can be separated more quickly.

The first electrophoresis is completed at normal temperature, or the electrophoresis buffer solution is cooled to below 8 ℃ before electrophoresis, and then electrophoresis is performed. During the second time of electrophoresis, before the electrophoresis, the electrophoresis buffer solution is cooled to 3-5 ℃, then the electrophoresis is carried out, pulse type square waves or oblique square waves are used in the electrophoresis, and the temperature of the buffer solution is prevented from being greatly improved.

The voltage amplitude of the pulse voltage is 300V, the pulse is a square wave or an oblique square wave, each pulse lasts for 10-15 seconds, then the next pulse is carried out after 10-15 seconds of interval, the duration of one pulse and the duration of the subsequent interval are a period, and the total duration of a plurality of periods is 1-2 minutes.

The specific structure of the first electrophoresis and the second electrophoresis is described below, using a dedicated apparatus.

the special equipment for the first electrophoresis is shown in figures 1-9, and the structure is as follows: including end box 1, electrophoresis box 10 and upper cover 28, a plurality of electrophoresis boxes are placed to the spaced in the end box, and the inside plate electrode 4 that sets up in every electrophoresis box both sides is connected with the binding post 2 electricity that sets up with the end box lateral wall of homonymy respectively, and this binding post is connected with the power electricity, lock upper cover on the end box.

in this embodiment, a bending plate 16 is disposed inside the side walls of the two sides of each electrophoresis cassette, one end of the bending plate located inside the electrophoresis cassette is bent downward to extend and form an electrode plate, and the other end 21 of the bending plate is electrically connected to the connection terminal. The preferred structure is: the outer surface of the side wall of the electrophoresis box aligned with the upper end of the bending plate is provided with a bulge 3, the lower end of the bulge is embedded into a groove 13 formed in the alignment of the bottom box, the end part of the other side of the bending plate extends downwards from the bottom surface of the bulge and contacts with the upper end of a connecting plate 15 arranged on the bottom surface of the groove, and the lower end 26 of the connecting plate is electrically connected with a wiring terminal.

The outer edge of a convex ring 23 at the lower end of the wiring terminal is provided with external threads and is meshed and connected with the surface of the side wall of the bottom box aligned with the wiring board, the end part 22 at the inner side of the wiring terminal is contacted with the wiring board, the end part at the outer side of the wiring terminal is provided with a wiring hole 24 and a fixing bolt 12 as shown in figure 6, the wiring hole is used for inserting the end part of a cable connected with a power supply, and the fixing bolt is used for fixing the end part of the cable in the end.

The wire insertion hole and the fixing bolt may also take a structure as shown in fig. 3, i.e., the fixing bolt 12 is disposed at the side of the end portion outside the terminal, and the wire insertion hole 24 is disposed at the end face of the end portion outside the terminal.

a plurality of partition plates 11 are arranged in the bottom box at intervals, the partitions divide the interior of the bottom box into a plurality of spaces 14, one electrophoresis box can be placed in each space, and different electrophoresis boxes are separated by the partition plates.

The middle part of the electrophoresis box is provided with a bulge 19, and two vertical plates 17 are respectively arranged at two edges of the bulge, which are close to two sides of the electrophoresis box; the areas of the electrophoresis cassette at the two sides of the bulge are polar plate areas 7 and 9, the area between two vertical plates at the upper end of the bulge is a gel tray area 5, and a gel tray 20 is placed in the gel tray area.

the electrode plate structure inside the electrophoresis cassette is shown in fig. 7, the upper end of the electrode plate structure extends into the insulation block 6 arranged opposite to the side wall of the electrophoresis cassette and is integrally connected with the bending plate, and the lower end of the electrode plate structure is arranged into a transverse bending structure 27.

the upper cover is provided with a plurality of temperature sensors 18 which are aligned with each electrophoresis box, the lower end of each temperature sensor extends below the liquid level of the buffer solution in the gel tray area, the output ends of the plurality of temperature sensors are connected with the wire row 29 which is arranged on the upper cover, all the wire rows are connected with the socket 30 which is arranged on the upper cover, a plug can be inserted into the socket, and the plug is connected with equipment which can receive and display data through a cable, such as an industrial personal computer, a thermometer, a computer and the like. The temperature sensor is used for detecting the temperature of the buffer solution in the gel tray area, the problem that the temperature of the buffer solution is too high due to the fact that the electrode plate is electrified is avoided, and when the temperature of the buffer solution is too high, the electrode plate is disconnected with the power supply.

The inner edge of the upper cover is provided with a convex rib 31 or a convex block at intervals, and the convex rib or the convex block at intervals is pressed on the upper end surface 8 of the bottom box after the upper cover is buckled on the bottom box. The lower end edge 32 of the upper cover shields the terminal.

the bottom box, the upper cover and the electrophoresis box are made of transparent insulating materials, the bending plate is made of platinum materials, and the wiring board and the wiring terminal are made of copper materials.

the use of this embodiment is shown in fig. 9: only two electrophoresis cassettes are used, placed in the two spaces at the leftmost side of the bottom cassette.

all the wiring terminals are connected with a power supply through cables, a plug is inserted into the socket, and the other end of the plug is connected with the thermometer.

1. Agarose gel trays were placed into the gel tray areas of the electrophoresis cassettes and the DNA samples were placed, two agarose gel trays were placed in the gel tray area in each electrophoresis cassette.

2. The first running buffer 25 was poured in, and then the power was turned on to apply power to the two electrode plates.

3. After a period of time, the power supply is cut off, the gel tray is taken out for cutting, the DNA gel block needing further separation is taken out for subsequent treatment.

the special equipment for the second electrophoresis is shown in fig. 10 to 21, and includes two power-taking modes, the first mode is a compression mode, and the second mode is a sliding mode, which are described below.

the first embodiment is shown in fig. 10 to 15, and has a structure that: including cooling box 1, electrophoresis box 10 and upper cover 28, set up at least one space 14 in the cooling box, place an electrophoresis box in every space, set up a plurality of mutual isolation's electrophoresis cell 34 in the electrophoresis box, the plate electrode 4 that every electrophoresis cell both sides set up realizes the electricity with the binding post 2 that the cooling box lateral wall of homonymy set up respectively through the mode of crimping to be connected, this binding post is connected with the power electricity, the lock upper cover on the cooling box, remain clearance 39 between the space of electrophoresis box and cooling box, this clearance communicates with coolant inlet 36 and the coolant export 35 that cooling box surface set up respectively.

in this embodiment, set up a plurality of baffles 33 in the electrophoresis box, these a plurality of baffles separate the electrophoresis box for a plurality of electrophoresis cell in, electrophoresis box bottom plate in every electrophoresis cell all is provided with arch 19, and two border departments that this arch is close to the electrophoresis cell both sides set up a riser 17 respectively, are gel stripping and slicing region 5 between two risers on every arch, and the electrophoresis cell of protruding both sides is electrode plate region 7, 9, places gel stripping and slicing 37 in gel stripping and slicing region.

The inside board 16 that bends that all sets up of electrophoresis box both sides lateral wall, the board that bends is located one side in the electrophoresis box and is provided with the tip that extends to the electrophoresis tank of bending downwards the same with electrophoresis cell quantity and forms electrode plate 4, and the other side tip of the board of bending is connected with binding post electricity. The upper end of the inner surface of the electrophoresis box is provided with a transverse insulating block 6, the bending plate is integrally connected with a transverse conductive plate in the insulating block, and the bottom surface of the conductive plate is integrally provided with the plurality of electrode plates. The preferred scheme is as follows: the outer surface of the side wall of the electrophoresis box which is opposite to the upper end of the bending plate is provided with a protrusion 3, the lower end of the protrusion is embedded into a groove 13 which is opposite to the cooling box, the end part 21 of the other side of the bending plate extends downwards from the bottom surface of the protrusion and is in compression joint with the upper end of a connecting plate 15 which is arranged on the bottom surface of the groove, and the lower end 26 of the connecting plate is electrically connected with the wiring terminal.

the outer edge of the convex ring 23 at the lower end of the wiring terminal is provided with external threads and is meshed and connected with the surface of the side wall of the cooling box aligned with the wiring board, the end part 22 at the inner side of the wiring terminal is contacted with the wiring board, the end part at the outer side of the wiring terminal is provided with a wiring hole 24 and a fixing bolt 12 as shown in figure 12, the wiring hole is used for inserting the end part of a cable connected with a power supply, and the fixing bolt is used for fixing the end part of the cable in the end.

at least one sealing ring 38 is arranged at the upper end of the outer edge of the electrophoresis box from top to bottom, two sealing rings are arranged up and down in the figure, the two sealing rings can be sleeved in a sealing ring groove arranged at the outer edge of the electrophoresis box, not only can the gap below be sealed, but also the electrophoresis box is stably embedded into the cooling box through friction.

The outer edge of the electrophoresis box below the sealing ring is provided with a recess 42, the inner edge of the cooling box opposite to the recess is provided with a recess 43, and the gap comprises two recesses, a gap 40 between the bottom surface of the electrophoresis box and the inner bottom surface of the cooling box and a space 41 in the protrusion. The lower end of one side surface of the cooling box is provided with a refrigerant inlet, the upper end of the other side surface of the cooling box is provided with a refrigerant outlet, and after the refrigerant enters the gap, the outer edge and the bottom surface of the electrophoresis box are soaked in the refrigerant.

The cooling box, the electrophoresis box and the upper cover are made of transparent insulating materials, the bending plate, the conductive plate and the electrode plate are made of platinum materials integrally, and the wiring terminal is made of copper materials.

As shown in fig. 11, the electrophoresis chamber is divided into four electrophoresis tanks, each of which has a different capacity, and is 1 ml, 2 ml, 5 ml and 10 ml from the rightmost side to the leftmost side. The refrigerant can be gas or liquid, and the gas can be cold air cooled by a compressor, or ice-salt mixture (the particles of ice are small).

The upper cover is directly buckled on the cooling box as shown in figure 14, and the electrophoresis box is not provided with a plug and a socket in the prior art, so that the upper cover is convenient to install and remove.

The use of this embodiment is shown in fig. 11: only one electrophoresis box is used, and the inside of the electrophoresis box is divided into four electrophoresis pools with different volumes. And connecting all the connecting terminals with a power supply through cables. The refrigerant is ice salt mixture. The third 5 ml from the right in FIG. 11 was selected (buffer was also added to the other electrophoresis cells).

1. Agarose gel sections were placed into the gel section area of the electrophoresis cell.

2. pouring buffer solution 25 for the second electrophoresis, introducing ice salt mixture from a refrigerant inlet through a water pump, and starting to electrify the electrode plate when the temperature of the buffer solution is measured to be 5 ℃.

3. After a period of time, the power is turned off, the buffer is removed with a pipette, and the DNA fragments are further separated by adsorption.

the second embodiment is shown in fig. 16 to 21, and has a structure that: including cooling box 1, electrophoresis box 10 and upper cover 28, place at least one electrophoresis box in the cooling box, set up a plurality of mutual isolation's electrophoresis cell 34 in the electrophoresis box, the plate electrode 4 that every electrophoresis cell both sides set up realizes the electricity with the binding post 2 that the cooling box lateral wall of homonymy set up respectively through gliding mode and is connected, this binding post is connected with the power electricity, the lock upper cover on the cooling box, remain clearance 39 between electrophoresis box and the cooling box, this clearance respectively with cooling box surface set up coolant import 36 and coolant export intercommunication 35.

In this embodiment, set up a plurality of baffles 33 in the electrophoresis box, these a plurality of baffles separate the electrophoresis box for a plurality of electrophoresis cell in, electrophoresis box bottom plate in every electrophoresis cell all is provided with arch 19, and two border departments that this arch is close to the electrophoresis cell both sides set up a riser 17 respectively, are gel stripping and slicing region 5 between two risers on every arch, and the electrophoresis cell of protruding both sides is electrode plate region 7, 9, places gel stripping and slicing 37 in gel stripping and slicing region.

The inside board 16 that bends that all sets up of electrophoresis box both sides lateral wall, the board that bends is located one side in the electrophoresis box and is provided with the tip that extends to the electrophoresis tank of bending downwards the same with electrophoresis cell quantity and forms electrode plate 4, and the other side tip of the board of bending is connected with binding post electricity. The upper end of the inner surface of the electrophoresis box is provided with a transverse insulating strip 6, the bending plate is integrally connected with a transverse conductive plate in the insulating strip, and the bottom surface of the conductive plate is integrally provided with the plurality of electrode plates. The preferred scheme is as follows: the other side end of the bending plate is provided with a connecting contact point 46, the outer side surface of the connecting contact point is provided with an arc shape, and the arc shape protrudes to the outer side of the side wall of the electrophoresis box and is in sliding contact with a connecting terminal arranged in a contraposition with the cooling box.

The outer edge of a convex ring 23 at the lower end of the wiring terminal is provided with external threads and is meshed and connected with the surface of the side wall of the cooling box aligned with the connecting contact point, the end part 22 at the inner side of the wiring terminal is contacted with the arc-shaped surface of the connecting contact point, the end part at the outer side of the wiring terminal is provided with a wiring hole 24 and a fixing bolt 12 as shown in fig. 19, the wiring hole is used for inserting the end part of a cable connected with a power supply, and the fixing bolt is used for fixing the end part of the cable in.

At least one sealing ring 38 is arranged at the upper end of the outer edge of the electrophoresis box from top to bottom, two sealing rings are arranged up and down in the figure, the two sealing rings can be sleeved in a sealing ring groove arranged at the outer edge of the electrophoresis box, not only can the gap below be sealed, but also the electrophoresis box is stably embedded into the cooling box through friction.

the outer edge of the electrophoresis box below the sealing ring is provided with a recess 42, and the gap comprises the recess, a gap 40 between the bottom surface of the electrophoresis box and the inner bottom surface of the cooling box and a space 41 inside the protrusion. The lower end of one side surface of the cooling box is provided with a refrigerant inlet, the upper end of the other side surface of the cooling box is provided with a refrigerant outlet, and after the refrigerant enters the gap, the outer edge and the bottom surface of the electrophoresis box are soaked in the refrigerant.

The cooling box, the electrophoresis box and the upper cover are made of transparent insulating materials, the bending plate, the conductive plate and the electrode plate are made of platinum materials integrally, and the wiring terminal is made of copper materials. The electrophoresis cassette is provided with a ridge or spaced projections 44 at the upper end of its outer edge to enable the cassette to be stably positioned on the upper end face 45 of the cooling cassette. The upper cover is directly buckled on the cooling box as shown in figure 20, and the electrophoresis box is not provided with a plug and a socket in the prior art, so that the upper cover is convenient to install and remove.

The internal division and use of the electrophoresis cassette are the same as those of the first embodiment.

after two times of electrophoresis, the buffer solution containing the DNA molecule fragments is removed and is subjected to adsorption treatment, and the electrophoresis box needs to be cleaned so as to be convenient for use in the next test. Because the electrophoresis chamber is divided into a plurality of electrophoresis chambers with different volumes, a better cleaning structure is needed to replace the conventional common manner of hand washing and soaking. The concrete structure is as follows:

as shown in fig. 22 to 26, the DNA electrophoresis cleaning device comprises a cleaning box 56, an end cover 55 and a transverse movement module, wherein the end cover is buckled at an opening at the upper end of the cleaning box, the transverse movement module is transversely arranged on the upper end surface of the end cover, the lower end of a liquid outlet pipe 53 arranged on the transverse movement module extends into the cleaning box, a plurality of micro-spray holes 69 are arranged at the lower end of the liquid outlet pipe, the liquid outlet pipe is communicated with a liquid outlet of a liquid pump, a liquid inlet 57 of the pump is communicated with a cleaning liquid source, and a plurality of DNA electrophoresis boxes 10 are transversely arranged at the bottom in the cleaning box.

In this embodiment, the washing solution 70 placed in the washing cassette soaks the DNA electrophoresis cassette. The transverse movement module drives the liquid outlet pipe to transversely move, and cleaning liquid sprayed by the micro-spray holes is used for spraying and cleaning the DNA electrophoresis box.

Above-mentioned liquid pump can set up on the workstation of wasing the box side, by the hose and the intercommunication of above-mentioned drain pipe upper end of its liquid outlet, also can adopt miniaturized liquid pump, is about to the liquid pump and directly sets up on the lateral motion module, and concrete structure is:

The transverse motion module is a linear motor, a transverse boss 50 is arranged on the upper end face of the end cover, a bottom plate 49 of the linear motor is installed on the boss through a bolt 58, a guide rail 48 is arranged on the bottom plate, a motion plate 51 is arranged on a slide block 54 which is arranged on the guide rail in a sliding mode, a liquid pump is arranged on the motion plate, and a liquid outlet pipe communicated with a liquid outlet of the liquid pump extends into the cleaning box from a transverse long hole 59 arranged on the boss and a transverse long hole 60 arranged in an aligned mode of the end cover. The bottom plate is provided with stoppers 47 at both sides as shown in fig. 22. The liquid outlet pipe can be of a two-section structure, namely, the lower end of one section connected with a liquid outlet of the liquid pump extends into the lower part of the end cover, and the other end of the liquid outlet pipe is connected with the lower end in a meshing mode, so that the liquid outlet pipe is convenient to clean or replace.

At the lower end of the outlet pipe is a longitudinal arc tube 68 as shown in fig. 24, the arc surface of which faces the bottom of the cleaning box and on which a plurality of micro-spray holes are provided.

A support 64 is arranged at the bottom in the cleaning box, the upper end face of the support is provided with transverse spaced beams 61, and a DNA electrophoresis box is arranged between the adjacent beams with two larger gaps. Between the two adjacent beams of the smaller gap are liquid passing ports 62 which communicate between the underside of the rack and the upper side of the rack, the lower ends 63 of the two sides of the rack as shown in fig. 25 resting against the inside surfaces of the side walls of the wash box and supporting the entire rack.

A gap 67 is reserved between the bottom surface of the DNA electrophoresis cassette and a bottom plate 66 of the washing cassette, a liquid discharge port 65 is provided on the side wall of the washing cassette on either side of the gap, and a valve is provided on the liquid discharge port.

and a heating rod 71 is arranged on any side of the cleaning box and on the side wall close to the bottom plate of the cleaning box, and the heating rod is connected with a power supply through a wiring terminal 73.

A liquid level probe 74 and a temperature sensor 72 are arranged in the cleaning box, and the output end of the liquid level probe, the output end of the temperature sensor, the control end of the liquid pump, the control end of the valve, the control end of the linear motor and the control end of the heating rod are all connected with an input/output interface of a control module. The control module takes a CPU as a core and is matched with various auxiliary circuits to complete data input, control instruction output and information storage.

The use process of this washing structure is:

Clean racks are placed in the washing box, and then the DNA electrophoresis box shown in FIG. 5, four in the figure, is placed in a larger gap. The supports with different specifications can be used for effectively supporting the DNA electrophoresis boxes with different sizes.

1. and starting a liquid pump, opening an end cover, adding a cleaning solution containing enzyme, soaking the DNA electrophoresis box, and buckling the end cover to ensure that the arc-shaped pipe extends below the liquid level of the cleaning solution.

After soaking for a period of time (2-3 minutes at normal temperature), the liquid pump is started to enable the micro-spray holes to spray cleaning liquid at a slow speed, and the valve of the liquid discharge port is properly opened to a certain opening degree so as to prevent the cleaning liquid from overflowing after the cleaning box is filled with the cleaning liquid. And the liquid spray of the micro-spray holes disturbs the cleaning liquid in the cleaning box, so that the cleaning effect is improved, and the soaking is completed for 5-10 minutes. In the process, the liquid level probe detects the liquid level height of the cleaning liquid at any time, and the control module automatically controls the opening of the valve and the flow of the liquid pump.

2. After soaking, the valve of the liquid discharge port is completely opened, and the cleaning liquid in the cleaning box is discharged (at the moment, the cleaning liquid is remained in the electrophoresis box, but the subsequent cleaning is not influenced because the volume of the electrophoresis box is smaller).

3. Starting the linear motor and the liquid pump, enabling the linear motor to transversely reciprocate, enabling the liquid pump to continuously suck clean cleaning liquid (such as water) from a cleaning liquid source, then spraying the cleaning liquid into the cleaning box and the electrophoresis box through the liquid outlet pipe and the micro-spraying holes, and stopping the linear motor and the liquid pump after a period of time (5-10 minutes) to finish spraying. In the process, the liquid level probe detects the liquid level height of the cleaning liquid at any time, and the control module automatically controls the opening of the valve and the flow of the liquid pump.

4. After the spray washing is finished, the valve is opened to the maximum opening degree, and liquid in the washing box is discharged. And opening the end cover, and adding a cleaning solution containing an enzyme inactivation reagent into the cleaning box. And the end cover is buckled, the linear motor is started and reciprocates, and clean cleaning liquid is sprayed at a low speed so as to disturb the liquid in the cleaning box. And (5) completing enzyme inactivation after soaking for a period of time, and taking out the electrophoresis cassette.

in addition to the enzyme inactivation by the addition of reagents, heating may also be used, in particular:

4. After the spray washing is finished, the valve is opened to the maximum opening degree, and liquid in the washing box is discharged. The liquid pump sucks clean water (cleaning liquid), then the heating rod starts to heat, the temperature sensor detects the liquid, the liquid in the cleaning box keeps a period of high temperature (80-100 ℃ for 10-15 minutes), then enzyme inactivation is completed, and the electrophoresis box is taken out.

in the method, the gel blocks obtained by the first electrophoresis are taken out and subjected to the second electrophoresis in the buffer solution, so that the DNA molecular fragments move in the buffer solution under the action of an electric field, and then the separation and purification of the DNA molecular fragments are carried out by an adsorption column or magnetic beads, the purity of the collected DNA molecular fragments is improved by 5-10% compared with that of the traditional method, and the separation and purification time is saved by more than 50% compared with that of the traditional method.