CN211713077U

CN211713077U - Plasmid extraction device

Info

Publication number: CN211713077U
Application number: CN201922193624.0U
Authority: CN
Inventors: 孙相鑫; 魏宏泉
Original assignee: Tuoyuan Onebio Technology Co ltd
Current assignee: Tuoyuan Onebio Technology Co ltd
Priority date: 2019-12-09
Filing date: 2019-12-09
Publication date: 2020-10-20
Anticipated expiration: 2029-12-09

Abstract

The application discloses plasmid extraction element, including memory storage body, reaction chamber and purification room, it deposits the chamber to have seted up 7 at least on the memory storage body, be provided with filter screen and filter membrane in the reaction chamber, filter screen and filter membrane separate into the reaction chamber independent and the first chamber that separates of intercommunication, second separate chamber and third and separate the chamber, the both sides of filter screen are first separate chamber and second respectively and separate the chamber, the both sides of filter membrane are the second respectively and separate chamber and third and separate the chamber, be provided with the GF membrane in the purification chamber, at least 3 deposit the chamber with the second separates the chamber and meets, and at least 4 deposit chamber and reaction chamber looks UNICOM, the third separate the chamber with reaction chamber looks UNICOM. The utility model discloses following beneficial effect has: this device can accomplish the extraction purification in-process of plasmid fast, convenient operation.

Description

Plasmid extraction device

Technical Field

The utility model relates to a biochemistry consumptive material field especially relates to a plasmid extraction element.

Background

The plasmid is a very important vector in genetic engineering, and the existing extraction method of the plasmid can be roughly divided into two steps, namely, the first step of cracking and the second step of purification, wherein the step of cracking can be divided into four small steps, the first small step is to carry out impurity removal pretreatment on bacterial liquid, the second small step is to add a reagent No. 1 (50mM glucose/25 mM Tris-HCl/10mM EDTA, pH 8.0), the third small step is to add a reagent No. 2 (0.2N NaOH/1% SDS), the fourth small step is to add a reagent No. 3 (3M potassium acetate/2M acetic acid), and after the four small steps are completed, supernatant is obtained by filtration.

After plasmid supernatant is obtained by first-step cracking, plasmids are adsorbed by a GF membrane, and then plasmid DNA is obtained by two times of washing and one time of elution.

Above-mentioned in-process needs use 7 kinds of at least reagents and multiple instrument, and the operation needs to use devices such as centrifuging tube, pipettor and centrifuge repeatedly, and the operation is very loaded down with trivial details.

SUMMERY OF THE UTILITY MODEL

The utility model aims at the above problem, a plasmid extraction device is proposed.

The utility model adopts the following technical scheme:

the utility model provides a plasmid extraction element, includes storage body, reaction chamber and purification room, it deposits the chamber to have seted up 7 at least on the storage body, be provided with filter screen and filter membrane in the reaction chamber, filter screen and filter membrane separate into first chamber, second chamber and the third chamber that separates of independent and intercommunication with the reaction chamber, the both sides of filter screen are first chamber and the second chamber that separates respectively, the both sides of filter membrane are the second respectively and separate chamber and third chamber, the second separates the intracavity activity and is provided with the rotor, be provided with the GF membrane in the purification chamber, at least 3 deposit the chamber with the second separates the chamber and meets, and at least 4 deposit the chamber and purify room looks UNICOM, the third separate the chamber with reaction chamber looks UNICOM.

The working process of the device is as follows, firstly, the number 1 reagent, the number 2 reagent, the number 3 reagent, the BL balancing liquid, the buffer WA, the buffer WB and the TE buffer solution are stored in the storage cavity on the storage body, and the working process is as follows

The first step is as follows: the bacteria culture solution is poured into the first separation cavity of the reaction chamber, the bacteria solution containing bacteria enters the second separation cavity, the bacteria are intercepted in the second separation cavity under the action of the filter membrane, the reaction chamber and the purification chamber are both connected with a vacuum pump, and the vacuum pump is used for vacuumizing the reaction chamber and the purification chamber and providing power for the liquid flow in the reaction chamber and the purification chamber.

The second step is that: add reagent suspension thallus No. 1 in proper order, reagent schizolysis thallus No. 2, No. 3 reagent deposits protein and genome DNA, contain the plasmid in the supernatant, the supernatant that contains the plasmid gets into the purification room behind the filtering action of filter membrane, the rotor rotation stirring liquid in this in-process second compartment (the rotor can be a magnet, also can be other rotating-structure realizes), the filter screen can play the fixed action to the rotor simultaneously, prevent that the rotor from rolling out the reaction chamber, this in-process vacuum pump is to the reaction chamber evacuation, the power of the liquid flow in the reaction chamber has been given.

The third step: before the supernatant enters the purification chamber, BL equilibrium liquid is added into the purification chamber, the BL equilibrium liquid is used for washing a GF membrane, then the supernatant is added, plasmids in the supernatant are adsorbed on the GF membrane, then buffer WA and buffer WB are sequentially added for washing, TE buffer solution is added after washing is finished, the plasmids are eluted and stored in the TE buffer solution, and a vacuum pump vacuumizes a centrifugal tube chamber connected with the purification chamber in the process, so that the power for liquid flowing in the purification chamber is provided.

This device can accomplish the extraction purification of plasmid fully automatically fast, convenient operation.

It is further noted that the present device can also be used to extract other nucleic acids.

Optionally, the rotor includes mounting disc, rolling disc and rotation post, rotation post fixed mounting is on the rolling disc, the rolling disc is installed on the mounting disc, mounting disc fixed mounting is in the second compartment intracavity.

This is an implementation manner of the rotor, and since the second compartment needs to be filled with reagent No. 1, reagent No. 2 and reagent No. 3 for reaction, the stirring structure is provided to accelerate the reaction. The concrete post that rotates can be the post of iron or contain the post of magnet, sets up magnet just so and can drive to rotate the post outside the reaction chamber and rotate, rotates the post and rotates and can stir liquid, the going on of reaction with higher speed.

Optionally, the reaction chamber is provided with three liquid inlets, a waste discharge port and liquid outlets, the three liquid inlets are directly communicated with the second separation cavity, the three liquid inlets are communicated with the 3 storage cavities on the storage body through guide pipes, and the liquid outlets are communicated with the purification chamber.

The liquid inlet is a passage for the reagent No. 1, the reagent No. 2 and the reagent No. 3 to enter the second separation chamber, the liquid outlet is used for discharging waste liquid generated in the reaction process, and the liquid outlet is used for a passage for supernatant liquid (containing plasmids) obtained by the reaction to leave the reaction chamber. The waste outlet is the passage for waste liquid to leave the reaction chamber.

Optionally, the membrane bioreactor further comprises a first clamping ring, a second clamping ring and a clamping block, wherein the first clamping ring is clamped in the reaction chamber, the clamping block is clamped on the first clamping ring, and the second clamping ring is used for clamping the filter membrane between the second clamping ring and the clamping block.

Optionally, the reaction chamber includes a tank body and a tank cover, one end of the tank cover is rotatably matched with the tank body, and the other end of the tank cover is in snap fit with the tank body.

Optionally, the GF membrane clamping device further comprises a first clamping ring, a second clamping ring and a clamping block, wherein the first clamping ring is clamped and fixed in the purification chamber, the clamping block is clamped on the first clamping ring, and the second clamping ring, the first clamping ring and the clamping block are used for clamping the GF membrane under the combined action.

Optionally, be provided with feed inlet, discharge gate and waste material row mouth on the purification room, the feed inlet has a plurality ofly, the feed inlet is used for depositing the chamber with continuous.

The feed inlet is a channel for supernatant, buffer WA, buffer WB and TE buffer solution to enter the purification chamber, the waste discharge outlet is a channel for waste liquid generated by reaction to leave the purification chamber, and after the reaction is finished, the TE buffer solution containing plasmid leaves the purification chamber from the discharge outlet.

Because waste liquid is generated in the extraction and purification processes, a waste liquid cylinder is arranged to collect the waste liquid flowing out of the reaction chamber and the purification chamber.

The purification chamber comprises a body and a cover, and the GF film is arranged in the body.

Optionally, the device further comprises a piston and a liquid outlet pipe, wherein the piston is movably and hermetically arranged in the storage cavity; the liquid outlet pipes are fixedly arranged on the storage body, each liquid outlet pipe is communicated with one storage cavity, and outlets of the liquid outlet pipes are arranged on the side pipe walls.

The purpose of the plunger is to squeeze the reagent out of the storage chamber by simply squeezing the plunger. The reagent leaves the storage chamber and enters the reaction chamber and the purification chamber.

Meanwhile, as the liquid outlet pipe is connected with hoses such as a silicone tube and the like during use, in order to prevent the silicone tube from being wound, the storage body is provided with a bayonet, and the silicone tube is clamped on the bayonet. Meanwhile, the outlet of the liquid outlet pipe is arranged on the wall of the side pipe, so that after the silicone tube is sleeved on the liquid outlet pipe, the silicone tube blocks the outlet, and liquid in the liquid outlet pipe cannot flow out from the outlet.

Optionally, still include the casing, the memory bank, reaction chamber and purification room all directly or indirectly set up on the casing, be provided with the draw-in groove on the casing, the draw-in groove is used for activity clamping centrifuging tube.

Because the TE buffer solution containing the plasmid is generally collected by a centrifuge tube for preservation, the above-mentioned card slot is designed to facilitate clamping of the centrifuge tube.

Optionally, the device further comprises an auxiliary collecting disc, wherein the auxiliary collecting disc is arranged on the shell and located on one side of the clamping groove, a channel is arranged in the auxiliary collecting disc, and one end of the channel is directly or indirectly communicated with the purification chamber.

Set up the effect of supplementary catch tray and make things convenient for the centrifuging tube to collect, after the centrifuging tube card was on the draw-in groove, the mouth of pipe of centrifuging tube aimed at the passageway of supplementary catch tray just, and the TE buffer solution that contains the plasmid gets into in the centrifuging tube just completely.

The utility model has the advantages that: this device can accomplish the extraction purification of plasmid fully automatically fast, convenient operation.

Description of the drawings:

FIG. 1 is a schematic diagram of a plasmid extraction apparatus;

FIG. 2 is a schematic view of the internal structure of a plasmid extraction apparatus;

FIG. 3 is a schematic diagram of a bank structure;

FIG. 4 is a schematic diagram of the structure of a reaction chamber;

FIG. 5 is a schematic diagram of the structure of a purification chamber;

FIG. 6 is a schematic diagram showing the positional relationship of the respective parts in the reaction chamber.

The figures are numbered: 1. the device comprises a shell, 101, a clamping groove, 2, an auxiliary collecting disc, 301, a tank body, 302, a tank cover, 303, a liquid inlet, 304, a liquid outlet, 305, a waste discharge port, 306, a filter screen, 307, a rotating disc, 308, a mounting disc, 309, a rotating column, 3010, a second clamping ring, 3011, a first clamping ring, 3012, a filter membrane, 3013, a clamping block, 3014, a first separation cavity, 3015, a second separation cavity, 3016, a third separation cavity, 401, a body, 402, a cover body, 403, a feed inlet, 404, a discharge port, 405, a waste discharge port, 406, a first clamping ring, 407, a second clamping ring, 408, a clamping block, 5, a storage body, 501, a storage cavity, 502, a liquid outlet tank, 503, a bayonet, 504, an outlet, 505, a piston, 6, a waste liquid tank, 7 and a shell plate.

The specific implementation mode is as follows:

the present invention will be described in detail with reference to the accompanying drawings.

As shown in fig. 1, fig. 2, and fig. 6, a plasmid extraction apparatus includes a storage body 5, a reaction chamber, and a purification chamber, wherein at least 7 storage cavities 501 are formed in the storage body 5, a filter screen 306 and a filter membrane 3012 are disposed in the reaction chamber, the filter screen 306 and the filter membrane 3012 separate the reaction chamber into a first compartment 3014, a second compartment 3015, and a third compartment 3016, which are independent and connected to each other, the first compartment 3014 and the second compartment 3015 are disposed on two sides of the filter screen 306, the second compartment 3015 and the third compartment 3016 are disposed on two sides of the filter membrane 3012, a rotor is movably disposed in the second compartment 3015, a GF membrane is disposed in the purification chamber, at least 3 storage cavities 501 are communicated with the second compartment 3015, at least 4 storage cavities 501 are communicated with the purification chamber, and the third compartment 3016 is communicated with the reaction chamber.

The working process of the device is as follows, firstly, the No. 1 reagent, the No. 2 reagent, the No. 3 reagent, the BL balancing solution, the buffer WA, the buffer WB and the TE buffer solution are stored in the storage cavity 501 on the storage body 5,

the working process is as follows

The first step is as follows: the bacteria culture solution is poured into the first separation cavity 3014 of the reaction chamber, the bacteria solution enters the second separation cavity 3015, bacteria are intercepted in the second separation cavity 3015 due to the action of the filter membrane 3012, the reaction chamber and the purification chamber are both connected with vacuum pumps, and the vacuum pumps are used for vacuumizing a downstream waste liquid chamber and providing power for liquid flowing in the reaction chamber and the purification chamber.

The second step is that: add reagent suspension thallus No. 1 in proper order, reagent schizolysis thallus No. 2, No. 3 reagent is precipitated protein and genome DNA, contain the plasmid in the supernatant, the supernatant that contains the plasmid gets into the purification room after filter effect of filter membrane 3012, the rotor rotation stirring liquid in this in-process second separate chamber 3015 (the rotor can be a magnet, also can be other rotating-structure and realize), filter screen 306 can play the fixed action to the rotor simultaneously, prevent that the rotor from rolling out the reaction chamber, this in-process vacuum pump is to the reaction chamber evacuation, the power of the liquid flow in the reaction chamber has been given.

The third step: before the supernatant enters the purification chamber, BL equilibrium liquid is added into the purification chamber, the GF membrane is washed, then the supernatant is added, the plasmid in the supernatant is adsorbed on the GF membrane, then the washing is carried out by using improper buffer WA and buffer WB in sequence, TE buffer solution is added after the washing is finished, the plasmid is eluted and stored in the TE buffer solution, and the vacuum pump vacuumizes the centrifuge tube in the process, so that the power for the liquid flow in the purification chamber is provided.

As shown in fig. 4, the rotor includes a mounting plate 308, a rotating plate 307, and a rotating post 309, the rotating post 309 is fixedly mounted on the rotating plate 307, the rotating plate 307 is mounted on the mounting plate 308, and the mounting plate 308 is fixedly mounted in the second compartment 3015.

The above structure is an implementation manner of the rotor, and since the second compartment 3015 needs to be filled with reagent No. 1, reagent No. 2, and reagent No. 3 for reaction, the stirring structure is provided to accelerate the reaction. The rotating column 309 can be an iron column or a column containing magnet, so that the magnet arranged outside the reaction chamber can drive the rotating column 309 to rotate, and the rotating column 309 rotates to stir liquid, thereby accelerating the reaction.

As shown in FIG. 4, the reaction chamber is provided with three inlet ports 303, a waste discharge port 305 and three outlet ports 304, the three inlet ports 303 are directly communicated with the second compartment 3015, the three inlet ports 303 are communicated with 3 storage cavities 501 on the storage body 5 through conduits, and the outlet ports 304 are communicated with the purification chamber.

The inlet port 303 is a passage for the reagent No. 1, the reagent No. 2 and the reagent No. 3 to enter the second compartment 3015, while the outlet port is used for discharging the waste liquid generated during the reaction, and the outlet port 304 is used for passing the supernatant (containing plasmid) obtained from the reaction out of the reaction chamber. The waste outlet 305 is the passage for waste liquid to leave the reaction chamber.

As shown in fig. 4, the membrane filtration apparatus further includes a first clamp ring 3011, a second clamp ring 3010, and a clamp block 3013, the first clamp ring 3011 is clamped in the reaction chamber, the clamp block 3013 is clamped on the first clamp ring 3011, and the second clamp ring 3010 is used to clamp the membrane filtration 3012 between the second clamp ring 3010 and the clamp block 3013.

As shown in fig. 4, the reaction chamber includes a tank 301 and a tank cover 302, one end of the tank cover 302 is rotatably engaged with the tank 301, and the other end of the tank cover 302 is snap-engaged with the tank 301.

The removable design of the canister lid 302 and canister lid 301 is to facilitate the addition of bacteria culture fluid to the first compartment 3014.

As shown in fig. 5, the device further comprises a first snap ring 406, a second snap ring 407 and a fixture block 408, wherein the first snap ring 406 is clamped and fixed in the purification chamber, the fixture block 408 is clamped and fixed on the first snap ring 406, and the second snap ring 407, the first snap ring 406 and the fixture block 408 are used for clamping the GF membrane under the combined action.

As shown in FIG. 5, the purification chamber is provided with a plurality of inlet ports 403, a plurality of outlet ports 404 and a plurality of waste discharge ports 405, wherein the inlet ports 403 are connected to the storage chamber 501 and the third compartment 3016.

The inlet 403 is the passage for supernatant, buffer WA, buffer WB and TE buffer to enter the purification chamber, the waste outlet 405 is the passage for waste liquid generated by the reaction to leave the purification chamber, and after the reaction is completed, TE buffer containing plasmid leaves the purification chamber from the outlet 404.

Since waste liquid is generated in the extraction and purification processes, a waste liquid cylinder 6 is provided for collecting the waste liquid flowing out from the reaction chamber and the purification chamber.

The purification chamber comprises a body 401 and a cover 402, and the GF film is disposed in the body 401.

As shown in fig. 3, the device further comprises a piston 505 and a liquid outlet pipe 502, wherein the piston 505 is movably and hermetically arranged in the storage cavity 501; the liquid outlet pipes 502 are fixedly arranged on the storage body 5, each liquid outlet pipe 502 is communicated with one storage cavity 501, and the outlets 504 of the liquid outlet pipes 502 are arranged on the side pipe wall.

The purpose of plunger 505 is to facilitate the addition of reagents into the reaction chamber and the purification chamber, and when reagents are added into storage chamber 501, the reagents can be extruded out of storage chamber 501 by squeezing plunger 505 by adding reagents into storage chamber 501 through outlet pipe 502.

Meanwhile, as the liquid outlet pipe 502 is connected with hoses such as a silicone tube and the like during use, in order to prevent the silicone tube from being wound, the storage body 5 is provided with a bayonet 503, and the silicone tube is clamped on the bayonet 503. Meanwhile, since the outlet 504 of the liquid outlet pipe 502 is formed in the side pipe wall, after the silicone tube is sleeved on the liquid outlet pipe 502, the silicone tube blocks the outlet 504, and the liquid in the liquid outlet pipe 502 cannot flow out from the outlet 504.

As shown in fig. 3, the centrifugal separation device further comprises a shell 1, wherein the storage body 5, the reaction chamber and the purification chamber are directly or indirectly arranged on the shell 1, a clamping groove 101 is formed in the shell 1, and the clamping groove 101 is used for clamping a centrifugal tube movably.

Since the TE buffer containing the plasmid is generally collected by a centrifuge tube for preservation, the above-mentioned card slot 101 is designed to facilitate the clamping of the centrifuge tube. The specific shell 1 is formed by fixedly splicing a plurality of shell plates 7 together.

As shown in the attached drawing 1, the device further comprises an auxiliary collecting tray 2, the auxiliary collecting tray 2 is arranged on the shell 1 and located on one side of the clamping groove 101, a channel is arranged in the auxiliary collecting tray 2, and one end of the channel is directly or indirectly communicated with the purification chamber.

Set up the effect of supplementary catch tray 2 and make things convenient for the centrifuging tube to collect, after the centrifuging tube card was on draw-in groove 101, the mouth of pipe of centrifuging tube aimed at supplementary catch tray 2's passageway just, and the TE buffer solution that contains the plasmid gets into in the centrifuging tube completely just.

The above only is the preferred embodiment of the present invention, not therefore the limit the patent protection scope of the present invention, all applications the equivalent structure transformation made by the contents of the specification and the drawings of the present invention is directly or indirectly applied to other related technical fields, and all the same principles are included in the protection scope of the present invention.

Claims

1. The utility model provides a plasmid extraction element, its characterized in that, includes storage body, reaction chamber and purification room, it deposits the chamber to have seted up 7 at least on the storage body, be provided with filter screen and filter membrane in the reaction chamber, filter screen and filter membrane separate into first chamber, second that separate the chamber and the third that communicate with the reaction chamber and separate the chamber, the both sides of filter screen are first chamber and second respectively and separate the chamber, the both sides of filter membrane are the second respectively and separate chamber and third and separate the chamber, the second separates the intracavity activity and is provided with the rotor, be provided with the GF membrane in the purification chamber, at least 3 deposit the chamber with the second separates the chamber and meets, at least 4 deposit the chamber and purify room looks UNICOM, the third separate the chamber with reaction chamber looks UNICOM.

2. The plasmid extraction device of claim 1 wherein the rotor comprises a mounting disk, a rotating disk, and a rotating post, the rotating post is fixedly mounted on the rotating disk, the rotating disk is mounted on the mounting disk, and the mounting disk is fixedly mounted in the second compartment.

3. The plasmid extraction apparatus as claimed in claim 1, wherein said reaction chamber is provided with three liquid inlets, a waste outlet and three liquid outlets, wherein said three liquid inlets are directly connected to said second compartment, said three liquid inlets are connected to 3 storage chambers of said storage body via conduits, and said liquid outlets are connected to said purification chamber.

4. The plasmid extraction device as claimed in claim 1, further comprising a first clamp ring, a second clamp ring and a clamp block, wherein the first clamp ring is clamped in the reaction chamber, the clamp block is clamped on the first clamp ring, and the second clamp ring is used for clamping the filter membrane between the second clamp ring and the clamp block.

5. The plasmid extraction apparatus as claimed in claim 1, wherein the reaction chamber comprises a pot body and a pot cover, one end of the pot cover is rotatably fitted with the pot body, and the other end of the pot cover is snap-fitted with the pot body.

6. The plasmid extraction device as claimed in claim 1, further comprising a first snap ring, a second snap ring and a fixture block, wherein the first snap ring is clamped and fixed in the purification chamber, the fixture block is clamped on the first snap ring, and the second snap ring, the first snap ring and the fixture block are used for clamping the GF membrane under the combined action.

7. The plasmid extraction device as claimed in claim 1, wherein the purification chamber is provided with a plurality of feed inlets, a plurality of discharge outlets and a waste discharge outlet, and the feed inlets are connected with the storage chamber.

8. The plasmid extraction device of claim 1 further comprising a piston and a liquid outlet pipe, wherein the piston is movably and hermetically disposed in the storage chamber; the liquid outlet pipes are fixedly arranged on the storage body, each liquid outlet pipe is communicated with one storage cavity, and outlets of the liquid outlet pipes are arranged on the side pipe walls.

9. The plasmid extraction device of claim 1 further comprising a housing, wherein the storage body, the reaction chamber and the purification chamber are directly or indirectly arranged on the housing, and the housing is provided with a clamping groove for the movable clamp centrifuge tube.

10. The plasmid extraction device according to claim 9, further comprising an auxiliary collection tray disposed on the housing and located at one side of the clamping groove, wherein a channel is disposed in the auxiliary collection tray, and one end of the channel is directly or indirectly communicated with the purification chamber.