CN115090230A

CN115090230A - 384 pore plate for primer synthesis and purification

Info

Publication number: CN115090230A
Application number: CN202210653245.9A
Authority: CN
Inventors: 喻明军; 王维坤; 崔康乐; 骆晓文; 陈健
Original assignee: General Biology Anhui Co ltd
Current assignee: General Biology Anhui Co ltd
Priority date: 2022-06-09
Filing date: 2022-06-09
Publication date: 2022-09-23
Anticipated expiration: 2042-06-09
Also published as: CN115090230B

Abstract

The invention discloses a 384-pore plate for primer synthesis and purification, which comprises a 384-pore plate body; each hole in the 384-hole plate body is designed into a gradually-changing circle from top to bottom, the bottoms of the gradually-changing circles are connected by a step and are in a low gradually-changing type semi-cone shape, the bottoms of the hole positions are designed into oblique openings, 6 cross beams are respectively arranged above and below the skirt edge sealing element, 3 cross beams are respectively arranged on the left and right of the skirt edge sealing element, and matched Frits carriers mainly comprise two types, one type is a Frits carrier with a diameter of 3.9mm (volume change rate: +/-0.2) and a thickness of 1-2 mm; the other is 2mm in diameter (the volume change rate is-0.3 mm) and 1-3mm in thickness; the invention provides a 384-pore plate which has good general performance of each synthesis instrument, high flexible application degree, stable quality of synthesized primers and compatibility of single-pore synthesis capacity and purification universality.

Description

384 pore plate for primer synthesis and purification

Technical Field

The invention relates to the technical field of primer synthesis, in particular to a 384-pore plate for primer synthesis and purification.

Background

The method for splicing the primers is a common method in whole gene synthesis, the gene synthesis demand is increased rapidly along with the rapid development of the whole molecular cloning industry, the production efficiency of the upstream primers is improved by carrying out micro-quantity and high-flux primer treatment, and the method is a target to be pursued by each gene synthesis factory. The 384-pore plate and the 96-pore plate are in the same area, and the number of pores is 4 times of the flux of the 96-pore plate, thereby not only improving the efficiency of batch treatment, but also realizing the microscale and reducing the production cost. Its mature application promotes the development and progress of gene synthesis primer production process.

Currently, the types of 384-pore plates on the market are gradually increased through the development of recent years, but the number of 384-pore plates on the market is not large due to the complex structure and high mold opening cost. And the primer treatment process needs to be considered in many aspects, such as Frits vector miniaturization, Frits loading capacity use width, maximum reagent amount loaded at one time, synthesis and purification consideration, and the like, and few 384-well plates with balanced final performance and good universality are available. Many problems exist because of the immature design or the injection molding process of the mould, etc., for example: some 384-hole plates can be normally synthesized in a platinum-768 model synthesis instrument, but the problems of air leakage and no drainage exist in a BLP or Leikun 768 model synthesis instrument; the problem that the reagent cannot effectively contact Frits because the carrier is deeply filled because more reagent doses are loaded at one time is solved; some injection molding processes are not considered, so that the problem that bubbles are easily formed in the plate to cause high defective products is solved; the problem of poor matching effect with Frits is caused by unreasonable structural design in some cases; some methods do not carefully summarize the fundamental difference of synthesis application of 384-well plates and 96-well plates, which causes the problem of high rejection rate of synthesized primers; the design of square orifices causes the problem that the space area of each hole is small, and unused hole sites cannot be sealed, so that the flexible application is poor.

Disclosure of Invention

The invention aims to solve the problems of the background technology, and provides a 384-well plate for primer synthesis and purification, which has good general performance of each synthesis instrument, high flexible application degree, stable quality of synthesized primers and compatibility of single-well synthesis capacity and purification.

The purpose of the invention can be realized by the following technical scheme:

a384-well plate for primer synthesis and purification comprises a 384-well plate body;

every hole in the 384 hole plate body is designed into a gradual change circle from top to bottom, the bottom of the gradual change circle is connected with a step and is a low gradual change type semi-cone, the bottom of the hole site is designed into a bevel opening, the upper part and the lower part of the skirt edge sealing piece are respectively provided with 6 cross beams, and the left part and the right part are respectively provided with 3 cross beams.

As a further scheme of the invention: the Frits carrier matched with the 384-pore plate body mainly has two types, one type is 3.9mm in diameter and 1-2mm in thickness; the other is 2mm in diameter and 1-3mm in thickness.

As a further scheme of the invention: the preparation process of the 384-pore plate body 1 comprises the following steps of:

step 1, raw materials: CPG loading capacity is 80-32umol/g, UHMW-PE molecular weight is 320 ten thousand, and mixing is carried out according to the mass ratio of 1: 14-28;

step 2, mixing uniformly: mixing the powder for 12 hours by using a V-shaped mixer;

step 3, die filling: coating the powder into a 384-hole plate die, compacting and supplementing the material through a pressure supplementing device, covering an upper cover plate and fixing;

and 4, sintering: preheating in a blast oven at 200 deg.C, placing at the top temperature, sintering for 17-19min, and cooling with water or naturally;

step 5, removing the mold: removing the upper die and the lower die, only leaving the middle die, and placing the Frits carrier in a round hole of the middle die;

step 6, filling: and (3) placing the intermediate die at a specified position of a filling machine, placing a 384-hole plate at the specified position of the filling machine, snapping a handle of the 384-hole plate, and filling Frits into corresponding positions of the 384-hole plate by 384 ejector pins.

As a further scheme of the invention: the press compensation device comprises an installation frame; be provided with compacting mechanism and feed supplement mechanism on the installing frame respectively, compacting mechanism is located the X axle direction of installing frame, and feed supplement mechanism is located the Y axle direction of installing frame, and compacting mechanism includes two compactors and first driving piece, and first driving piece is connected with the compactors, and feed supplement mechanism includes two feed supplements and second driving piece, and the second driving piece is connected with the feed supplement.

As a further scheme of the invention: connecting shafts are respectively arranged at two ends of a connecting plate of the compaction piece, a plurality of compaction blocks are uniformly arranged on the bottom surface of the connecting plate, one end, far away from the connecting plate, of each connecting shaft is connected with a limiting sleeve, the limiting sleeves are slidably sleeved on the frame of the mounting frame, and the two limiting sleeves are connected with the second driving piece.

As a further scheme of the invention: the spacing of the compacted blocks is matched with the hole sites in the Y-axis direction of the pore plate, and the number of the compacted blocks is equal to that of the hole sites of the pore plate.

As a further scheme of the invention: the top surface both ends cover of the flow distribution plate of benefit material spare is equipped with the gag lever post to with gag lever post sliding connection, the both ends of gag lever post set up respectively on the inner wall of installing frame, the top surface of flow distribution plate is provided with two material pipes, the bottom surface of flow distribution plate is provided with the discharge opening, discharge opening department installs the notes stub bar, two flow distribution plates are connected with first driving piece respectively.

As a further scheme of the invention: the space between the material injection heads is matched with the hole sites in the X-axis direction of the pore plate, and the number of the material injection heads is equal to that of the hole sites of the pore plate.

As a further scheme of the invention: the first driving piece and the second driving piece adopt a screw rod transmission mode.

The invention has the beneficial effects that:

(1) compared to the square well 384 plate: the matched Frits error tolerance rate is high due to the gradual design of the circular holes, thereby being beneficial to the batch production of Frits carriers and reducing the research and development cost and period of Frits; the spacing area between the circular holes is larger, the unused area can be effectively stuck and sealed by using aluminum foil paper, and the falling-off condition can not occur, so that the circular holes can be used without being full, and the flexible application capability of a 96-hole plate is realized; two types of Frits carriers with large diameter size difference can be really used, and the low porosity Frits synthesis and the high porosity Frits synthesis can be realized, so that the adaptability is wider; the device has no connecting structure of a rectangular cavity and a rotary cylindrical cavity, has small requirement on the spraying precision of the needle of the instrument, and the reagent with the deviation of +/-2 mm can also completely flow into Frits along the hole wall, so that the effective components fully react, the reaction effect is improved, and the synthetic mutation rate of the primer is reduced;

(2) compared to a flat bottom 384 plate: the inclined cutting bottom plate increases the opening area, is more beneficial to discharging and non-adhesion of high-pressure reagents, and cannot influence the next reaction because the reagent is not dried, so that the success rate of the reaction is improved, and the rejection rate is reduced;

(3) compared with 384 plates with unreasonable beam design: the structural design is more reasonable, the deformation amplitude is small after injection molding is finished, so that the device can be matched with instruments with small sealing ring diameters for use, and the general performances of various types of synthesizers and purifiers are high;

(4) compared to 384 plates, which are not properly dimensioned: the sizes of the product before and after the product is subjected to cold shrinkage after demolding are accurately evaluated, the size of the final product is more standard, the product has high universality with various types of synthetic instruments and purifying instruments, and the problems of difficulty in taking and placing and low fitting degree with a sealing ring are solved;

(5) compared to a deep-well 384-well plate: the reagent has short stroke, small gravity, low probability of forming bubbles, high efficiency of reagent reaction and reduction of the synthetic mutation rate of the primer;

(6) compared to 384 well plates that can be packed with a Frits volume: because the highest load capacity performance of CPG (Controlled-pore glass) is broken through in two years, the large-scale synthesis of Frits is miniaturized, the Frits with two sizes can be used for preparing 50-100nmol carriers, larger-volume Frits do not need to be filled, the reaction reagent amount has obvious advantages, and the requirement of batch and large-scale synthesis is smaller.

Drawings

The invention will be further described with reference to the accompanying drawings.

FIG. 1 is a schematic structural view of the present invention;

FIG. 2 is a schematic structural view of a 384 well plate body of the present invention;

FIG. 3 is a cross-sectional view of the invention A-A;

FIG. 4 is a schematic view of a hole site according to the present invention;

FIG. 5 is a schematic view of the press-repairing apparatus of the present invention;

FIG. 6 is a schematic view of the feed mechanism of the present invention;

fig. 7 is a schematic view of the construction of the compaction mechanism of the invention.

In the figure: 1. a 384-well plate body; 2. a pressure compensation device; 21. installing a frame; 22. a splitter plate; 23. a limiting rod; 24. a first motor; 25. a first lead screw; 26. injecting a material head; 27. a connecting plate; 28. compacting the block; 29. a connecting shaft; 210. a limiting sleeve; 211. a second motor; 212. a second lead screw; 213. a material pipe; 214. an elastic tube.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Referring to fig. 1 to 4, the present invention is a 384-well plate for primer synthesis and purification, including a 384-well plate body 1, wherein the 384-well plate body 1 can be highly matched with a plurality of cylindrical Frits synthesis vectors, filter elements, and purification vectors; each hole in the 384-hole plate body 1 is designed into a gradually-changing circle from top to bottom, the bottom of the gradually-changing circle is connected with a step and is a low gradually-changing type semi-cone, the bottom of each hole is designed into an oblique opening, the upper part and the lower part of a skirt edge sealing element are respectively provided with 6 cross beams, the left part and the right part are respectively provided with 3 cross beams, and matched Frits carriers mainly have two types, one type is a Frits carrier with the diameter of 3.9mm (the volume change rate is +/-0.2) and the thickness of 1-2 mm; the other is 2mm in diameter (the volume change rate is-0.3 mm) and 1-3mm in thickness;

example 2

The process for preparing the 50nmol 384-hole synthetic plate by matching the diameter of 4mm and the thickness Frits of 2mm comprises the following steps:

step 1, raw materials: CPG loading capacity is 80umol/g, UHMW-PE molecular weight is 320 ten thousand, and mixing is carried out according to a ratio of 1: 14;

step 2, uniformly mixing: mixing the powder for 12 hours by using a V-shaped mixer;

step 3, die filling: coating the powder into a 384-pore plate mold, compacting, vibrating by using an oscillator, supplementing the powder, compacting again, covering an upper cover plate and fixing;

step 5, form removal: removing the upper die and the lower die, only leaving the middle die, and placing the Frits carrier in a round hole of the middle die;

step 6, filling: placing the middle mould at a specified position of a filling machine, placing a 384-hole plate at the specified position of the filling machine, snapping a handle of the 384-hole plate, and filling Frits into corresponding positions of the 384-hole plate by 384 ejector pins;

step 7, checking: and manually checking whether the carrier is defective or skewed, manually removing unqualified products if the carrier is defective or skewed, supplementing the unqualified products, and checking again until the carrier is in compliance.

Example 3

The process for preparing the 178-hole composite plate with 5nmol specification by matching 2mm diameter and 2mm thickness Frits comprises the following steps:

step 1, raw materials: CPG loading capacity is 32umol/g, UHMW-PE molecular weight is 320 ten thousand, and mixing is carried out according to a ratio of 1: 28;

step 4, sintering: preheating in a blast oven at 200 deg.C, placing at the top temperature, sintering for 17-19min, and cooling with water or naturally;

step 6, filling: placing the middle mould at a specified position of a filling machine, placing a 384-hole plate at the specified position of the filling machine, setting a filling area, snapping a handle of the 384-hole plate, and filling Frits into corresponding positions of the 384-hole plate by 178 thimbles;

step 7, sealing: and (3) sealing the hole position without the carrier by using aluminum foil paper, and cutting the redundant part by using an art designer.

Example 4

Matching 4mm diameter by 2mm thickness carrier, making DMT 384-well purification plate process, including the following steps:

step 1, carrier source: outsourcing;

step 2, filling: a common worker plugs the carrier into the 384-hole plate, places the carrier in a designated area, breaks a handle of the 384-hole plate, and loads the carrier into corresponding positions of the 384-hole plate by 384 thimbles;

step 3, checking: and manually checking whether the carrier is defective or skewed, manually removing unqualified products if the carrier is defective or skewed, supplementing the unqualified products, and checking again until the carrier is in compliance.

Example 5

Referring to fig. 5-7, based on the above embodiments 2 and 3, a press compensation device 2 is adopted for mold filling;

the press-repairing device 2 comprises a mounting frame 21; the mounting frame 21 is respectively provided with a compacting mechanism and a material supplementing mechanism, the compacting mechanism is positioned in the X-axis direction of the mounting frame 21, the material supplementing mechanism is positioned in the Y-axis direction of the mounting frame 21, the compacting mechanism comprises two compacting pieces and a first driving piece, the first driving piece is connected with the compacting pieces, the two compacting pieces are driven to move mutually through the first driving piece, so that materials in the mold are compacted, the material supplementing mechanism comprises two material supplementing pieces and a second driving piece, the second driving piece is connected with the material supplementing pieces, the two material supplementing pieces are driven to move mutually through the second driving piece, and therefore a cavity in the mold is supplemented with materials;

the compacting piece comprises a connecting plate 27, compacting blocks 28, connecting shafts 29 and limiting sleeves 210, the connecting shafts 29 are arranged at two ends of the connecting plate 27 respectively, a plurality of compacting blocks 28 are uniformly arranged on the bottom surface of the connecting plate 27, the spacing of the compacting blocks 28 is matched with the hole positions of the 384-hole plate in the Y-axis direction, the number of the compacting blocks 28 is equal to that of the 384-hole plate, one end, far away from the connecting plate 27, of the connecting shaft 29 is connected with the limiting sleeves 210, the limiting sleeves 210 are slidably sleeved on the frame of the mounting frame 21, and the two limiting sleeves 210 are connected with the second driving piece; the compaction block 28 is connected with the connecting plate 27 through the elastic tube 214, and the elastic tube 214 is arranged, so that the compaction block 28 and the die can be elastically extruded, the die can be protected, and the compaction effect can be better;

the second driving piece comprises a second motor 211 and a second screw rod 212, the second motor 211 is arranged on the side wall of the mounting frame 21, the output end of the second motor 211 is connected with the second screw rod 212, the second screw rod 212 is rotatably mounted in the frame of the mounting frame 21, two ends of the second screw rod 212 are respectively sleeved with a mounting block and are connected with the limiting sleeve 210 through the mounting block, a sliding groove matched with the mounting block is formed in the frame of the mounting frame 21, and the threaded connection mode of the two sliding blocks and the second screw rod 212 is opposite;

when the die works, the second motor 211 is controlled to work to drive the second screw rod 212 to work, the compaction blocks 28 on the connecting plate 27 are driven to move mutually through the sliding block, the limiting sleeve 210 and the connecting shaft 29, and the mounting frame 21 is driven to integrally move downwards through the air cylinder, so that the compaction blocks 28 compact materials in the die according to the progress of every two rows;

the material supplementing part comprises a splitter plate 22, a limiting rod 23, material injection heads 26 and material pipes 213, wherein the limiting rod 23 is sleeved at two ends of the top surface of the splitter plate 22 and is in sliding connection with the limiting rod 23, two ends of the limiting rod 23 are respectively arranged on the inner wall of the mounting frame 21, the two material pipes 213 are arranged on the top surface of the splitter plate 22, a discharge hole is arranged on the bottom surface of the splitter plate 22, the material injection heads 26 are arranged at the discharge hole, the space between the material injection heads 26 is matched with the hole sites in the X-axis direction of the 384 pore plate, and the number of the material injection heads 26 is equal to that of the 384 pore plate; the two splitter plates 22 are respectively connected with the first driving piece;

the first driving piece comprises a first motor 24 and a first screw rod 25, the first motor 24 is arranged on the side wall of the mounting frame 21, the output end of the first motor 24 is connected with the first screw rod 25, the first screw rod 25 is rotatably arranged on the mounting frame 21, the first screw rod 25 is in threaded connection with the tops of the two shunting plates 22, and the threaded connection modes are opposite;

when the material feeding device works, the first motor 24 is controlled to work to drive the first screw rod 25 to work, the limiting rod 23 is used for driving the material injection heads 26 on the flow distribution plate 22 to move mutually, and the air cylinder is used for driving the mounting frame 21 to integrally move downwards, so that the compaction blocks 28 feed materials in the die according to the progress of every two rows;

therefore, the press repairing device 2 is highly matched with the 384-hole plate, so that the feeding speed to the mold is greatly increased, the compacting speed to the mold is greatly increased, the press repairing device and the 384-hole plate are matched with each other and work cooperatively, the press repairing device and the mould are of an integrated structure, and the press repairing device are shifted during working to ensure that each mechanism works normally and orderly, so that the forming efficiency of the 384-hole plate is improved.

While one embodiment of the present invention has been described in detail, the description is only a preferred embodiment of the present invention and should not be taken as limiting the scope of the invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.

Claims

1. A384-well plate for primer synthesis and purification, comprising a 384-well plate body (1);

each hole in the 384-hole plate body (1) is designed into a gradually-changing circle from top to bottom, the bottom of the gradually-changing circle is connected with a step and is a low gradually-changing type semi-cone, the bottom of the hole is designed into an oblique opening, the upper part and the lower part of a skirt seal piece are respectively provided with 6 cross beams, and the left part and the right part of the skirt seal piece are respectively provided with 3 cross beams.

2. The 384-well plate for primer synthesis and purification as claimed in claim 1, wherein the 384-well plate body (1) is matched with Frits vector of two types, one is 3.9mm in diameter and 1-2mm in thickness; the other is 2mm in diameter and 1-3mm in thickness.

3. The 384-well plate for primer synthesis and purification according to claim 1, wherein the 384-well plate body (1) is prepared by the following steps:

step 3, die filling: coating the powder into a 384-pore plate die, compacting and supplementing the material through a pressure supplementing device (2), covering an upper cover plate and fixing;

and 4, sintering: preheating in a blast oven at 200 deg.C, placing at the top temperature, sintering for 17-19min, taking out, and cooling with water or naturally;

4. The 384-well plate for primer synthesis and purification according to claim 3, wherein the press-fit device (2) comprises a mounting frame (21); be provided with compacting mechanism and feed supplement mechanism on installing frame (21) respectively, compacting mechanism is located the X axle direction of installing frame (21), and feed supplement mechanism is located the Y axle direction of installing frame (21), and compacting mechanism includes two compacts and first driving piece, and first driving piece is connected with the compacts, and feed supplement mechanism includes two feed supplement pieces and second driving piece, and the second driving piece is connected with the feed supplement piece.

5. The 384-well plate for primer synthesis and purification as claimed in claim 4, wherein the two ends of the connecting plate (27) of the compacting member are respectively provided with a connecting shaft (29), the bottom surface of the connecting plate (27) is uniformly provided with a plurality of compacting blocks (28), one end of the connecting shaft (29) far away from the connecting plate (27) is connected with a limiting sleeve (210), the limiting sleeve (210) is slidably sleeved on the frame of the mounting frame (21), and both limiting sleeves (210) are connected with the second driving member.

6. The 384-well plate for primer synthesis and purification according to claim 5, wherein the distance between the compacted lumps (28) is adapted to the number of wells on the 384-well plate in the Y-axis direction, and the number of compacted lumps (28) is equal to the number of wells on the 384-well plate.

7. The 384-hole plate for primer synthesis and purification as claimed in claim 6, wherein two ends of the top surface of the flow distribution plate (22) of the supplement member are sleeved with the limiting rod (23) and are slidably connected with the limiting rod (23), two ends of the limiting rod (23) are respectively disposed on the inner wall of the mounting frame (21), two material pipes (213) are disposed on the top surface of the flow distribution plate (22), a discharge hole is disposed on the bottom surface of the flow distribution plate (22), a material injection head (26) is installed at the discharge hole, and the two flow distribution plates (22) are respectively connected with the first driving member.

8. The 384-well plate for primer synthesis and purification according to claim 7, wherein the pitch of the filling heads (26) is adapted to the hole sites of the 384-well plate in the X-axis direction, and the number of the filling heads (26) is equal to the number of the hole sites of the 384-well plate.

9. The 384-well plate for primer synthesis and purification according to claim 8, wherein the first driving member and the second driving member are driven by a screw.