CN111979109A - Plasmid vector continuous cracking device - Google Patents
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Abstract
The invention discloses a plasmid vector continuous cracking device, wherein the plasmid vector continuous cracking device comprises a reaction vessel and a driving component; the reaction container comprises a first container, a second container, a third container and a fourth container, wherein the first container is used for containing heavy suspension turbid liquid comprising thalli and heavy suspension liquid, the second container is used for containing lysis liquid, the third container is used for containing neutralization liquid, and the fourth container is used for containing lysis turbid liquid comprising heavy suspension turbid liquid and lysis liquid; the driving assembly comprises a first driving pump, a second driving pump and a third driving pump, the first driving pump is used for pumping the heavy suspension turbid liquid in the first container to the fourth container, the second driving pump is used for pumping the lysate in the second container to the fourth container, and the third driving pump is used for pumping the lysis turbid liquid in the fourth container to the third container. The plasmid vector continuous cracking device ensures the stability of thallus cracking and reduces the time of thallus cracking.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a continuous cracking device for a plasmid carrier.
Background
The plasmid extraction by cracking the bacterial thallus is a common technique in the biotechnology field. In the conventional manual lysis method, an operator pours a resuspension solution into a container containing bacteria, resuspends the bacteria to form a resuspended turbid solution, then adds a lysis solution into the container to form a lysed turbid solution, and then rapidly rotates the container, during the rotation of the operator, the bacteria are continuously lysed by the lysis solution to release DeoxyriboNucleic Acid (DNA), Ribonucleic Acid (RNA), Host Cell Protein (HCP), Host Cell DNA (HCD), saccharides and other substances.
In order to ensure a good bacterial cell lysis effect, an operator needs to rotate the container at a certain speed within a certain time and change the rotation speed by observing the state of the lysis suspension in the container, if the operation is too violent, the genome DNA is damaged, so that the genome DNA cannot be effectively removed, and if the operation is too gentle, the bacterial cell lysis is insufficient, so that the total yield of the objective plasmid DNA is reduced. When the amount of the cells to be lysed is relatively large, the operator needs to perform the above operation several times. However, inevitable differences are generated in each manual operation, and the generated differences are large, so that the thallus cracking effect cannot be completely consistent in each manual operation; moreover, the operation of the thallus cracking is long in time consumption, and the consumed manpower and material resources are also high.
The above is only for the purpose of assisting understanding of the technical solutions of the present invention, and does not represent an admission that the above is the prior art.
Disclosure of Invention
The invention mainly aims to provide a continuous cracking device for a plasmid carrier, which aims to ensure the stability of thallus cracking and reduce the time of thallus cracking.
In order to achieve the above object, the present invention provides a continuous cracking apparatus for a plasmid carrier, comprising:
the reaction container comprises a first container, a second container, a third container and a fourth container, wherein the first container is used for containing a heavy suspension turbid liquid comprising thalli and a heavy suspension liquid, the second container is used for containing a lysis solution, the third container is used for containing a neutralization solution, and the fourth container is used for containing a lysis turbid liquid comprising the heavy suspension turbid liquid and the lysis solution; and
the driving assembly comprises a first driving pump, a second driving pump and a third driving pump, the first driving pump is used for pumping the heavy suspension turbid liquid in the first container into the fourth container, the second driving pump is used for pumping the lysis liquid in the second container into the fourth container, and the third driving pump is used for pumping the lysis turbid liquid in the fourth container into the third container.
In an embodiment, the reaction vessel further comprises a collection device for holding the neutralized turbid liquid comprising the lysed turbid liquid and the neutralized liquid, the drive assembly further comprises a fourth drive pump for pumping the lysed turbid liquid into the collection device, the fourth drive pump for pumping the neutralized liquid into the collection device.
In one embodiment, the plasmid vector continuous lysis device further comprises a first connecting pipe, a second connecting pipe, a third connecting pipe and a fourth connecting pipe, wherein the first connecting pipe is communicated with the first container and the fourth container, the second connecting pipe is communicated with the second container and the first connecting pipe, the third connecting pipe is communicated with the fourth container and the collection device, and the fourth connecting pipe is communicated with the third container and the third connecting pipe.
In one embodiment, the first, second and third actuation pumps are diaphragm pumps.
In one embodiment, the continuous plasmid vector lysis device further comprises a resuspension solution, a lysis solution and a neutralization solution, wherein the volume ratio of the resuspension solution to the lysis solution to the neutralization solution is 2: 4: 3.
in one embodiment, the resuspension comprises tris (hydroxymethyl) aminomethane-hydrochloric acid at a concentration of 50mmol/L, ethylenediaminetetraacetic acid at a concentration of 10mmol/L, and glucose at a concentration of 100 mmol/L.
In one embodiment, the lysis solution comprises sodium hydroxide and sodium dodecyl sulfate, the concentration of the sodium hydroxide is 300mmol/L, and the ratio of the weight to the volume of the sodium dodecyl sulfate is 1%.
In one embodiment, the neutralizing liquid comprises potassium acetate, and the concentration of the potassium acetate is 3 mol/L.
In one embodiment, the reaction vessel is a reaction barrel, a reaction tank, a reaction flask or a reaction tank.
In one embodiment, the continuous plasmid vector lysis device further comprises a stirring member disposed in the fourth container for stirring the lysis turbid liquid in the fourth container.
The continuous lysis device for the plasmid vector comprises a reaction container and a driving assembly, wherein the reaction container comprises a first container, a second container, a third container and a fourth container, and the driving assembly comprises a first driving pump, a second driving pump and a third driving pump; according to the plasmid vector continuous cracking device, the first container is used for containing the heavy suspension turbid liquid comprising the thalli and the heavy suspension liquid, the second container is used for containing the lysis liquid, the third container is used for containing the neutralization liquid, the first driving pump is used for pumping the heavy suspension turbid liquid in the first container to the fourth container, the second driving pump is used for pumping the lysis liquid in the second container to the fourth container, and the third driving pump is used for pumping the lysis turbid liquid in the fourth container to the third container.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.
FIG. 1 is a schematic structural diagram of a continuous plasmid vector lysis apparatus according to an embodiment of the present invention;
FIG. 2 is a schematic structural diagram of another embodiment of the continuous lysis apparatus for plasmid vector according to the present invention;
FIG. 3 is a graph showing the results of HCP content analysis of the filtrates from the first and second set of experiments;
FIG. 4 is a graph showing the results of HCD content analysis of the filtrates from the first and second set of experiments;
FIG. 5 is a graph of the results of an analysis of the supercoiled proportion of the concentrate from the first and second set of experiments;
FIG. 6 is a graph showing the result of HCP content analysis of the permeate of a conventional manual lysis and continuous lysis apparatus;
FIG. 7 is a graph showing the result of HCD content analysis of the filtrate of a conventional manual cracking and continuous cracking apparatus;
FIG. 8 is a graph showing the results of analysis of the supercoiled proportion of the concentrated solution of the conventional manual lysis and continuous lysis apparatus.
The reference numbers illustrate:
the implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
It should be noted that if the description of "first", "second", etc. is provided in the embodiment of the present invention, the description of "first", "second", etc. is only for descriptive purposes and is not to be construed as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, the meaning of "and/or" appearing throughout is to include three juxtapositions, exemplified by "A and/or B" including either scheme A, or scheme B, or a scheme in which both A and B are satisfied.
The invention provides a continuous cracking device for a plasmid carrier.
Referring to fig. 1 and 2, the plasmid vector continuous lysis apparatus 100 includes a reaction vessel 110 and a driving assembly 120; the reaction vessel 110 comprises a first vessel 111, a second vessel 112, a third vessel 113 and a fourth vessel 114, wherein the first vessel 111 is used for containing a resuspended turbid solution containing thalli and a resuspended solution, the second vessel 112 is used for containing a lysis solution, the third vessel 113 is used for containing a neutralization solution, and the fourth vessel 114 is used for containing a lysed turbid solution containing the resuspended turbid solution and the lysis solution; the driving assembly 120 includes a first driving pump 1211, a second driving pump 1212, and a third driving pump 1213, wherein the first driving pump 1211 is used for pumping the re-suspended turbid solution in the first container 111 into the fourth container 114, the second driving pump 1212 is used for pumping the lysis solution in the second container 112 into the fourth container 114, and the third driving pump 1213 is used for pumping the lysis turbid solution in the fourth container 114 into the third container 113.
In the embodiment of the present invention, the reaction container 110 has various structures, for example, the reaction container 110 may include a partition dividing the reaction container 110 into four sub-containers that are not communicated with each other, so as to contain the resuspended turbid solution, the lysate, the lysed turbid solution, and the neutralizing solution, respectively. The partition may be a partition plate or a partition groove. The reaction vessel 110 may be a reaction barrel, a reaction tank, a reaction flask or a reaction tank. The top ends of the first container 111, the second container 112, the third container 113 and the fourth container 114 are respectively provided with a first opening, a second opening, a third opening and a fourth opening. The first container 111 may further include a first top cover, and the first top cover is disposed at the first opening and used for opening or closing the first opening. The second container 112, the third container 113, and the fourth container 114 may further include a second top cover, a third top cover, and a fourth top cover, respectively, and the first top cover is referred to for the positions and functions of the second top cover, the third top cover, and the fourth top cover. In order to achieve a better continuous lysis effect of the bacteria, the reaction vessel 110 may further include a collecting device 115, and the collecting device 115 will be described in detail later and will not be described herein.
In the embodiment of the present invention, in order to facilitate the driving of the resuspended turbid liquid, the lysate and the lysed turbid liquid by the first driving pump 1211, the second driving pump 1212 and the third driving pump 1213, respectively, the first driving pump 1211, the second driving pump 1212 and the third driving pump 1213 are respectively disposed at the upper ends of the first container 111, the second container 112 and the fourth container 114. In order to achieve a better continuous lysis effect of the bacteria, the driving assembly 120 may further include a fourth driving pump 1214 and a connecting pipe 122, which will be described in detail later and will not be described herein again.
The continuous lysis apparatus 100 for plasmid vector of the present invention comprises a reaction vessel 110 and a driving assembly 120, wherein the reaction vessel 110 comprises a first vessel 111, a second vessel 112, a third vessel 113 and a fourth vessel 114, and the driving assembly comprises a first driving pump 1211, a second driving pump 1212 and a third driving pump 1213; according to the invention, the first container 111 is used for containing the resuspended turbid liquid comprising the thallus and the resuspended liquid, the second container 112 is used for containing the lysis liquid, the third container 113 is used for containing the neutralization liquid, the first driving pump 1211 is used for pumping the resuspended turbid liquid in the first container 111 to the fourth container 114, the second driving pump 1212 is used for pumping the lysis liquid in the second container 112 to the fourth container 114, and the third driving pump 1213 is used for pumping the lysed turbid liquid in the fourth container 114 to the third container 113, so that when an operator needs to lyse a large amount of thallus, the plasmid vector continuous thallus lysis device can continuously lyse a large amount of thallus, the thallus lysis stability is ensured, and the thallus lysis time is shortened.
The collection device 115 and the fourth drive pump 1214 are described below.
Referring to fig. 2, in an embodiment, the reaction container 110 further includes a collecting device 115, the collecting device 115 is used for containing the lysis turbid liquid and the neutralized turbid liquid, the driving assembly 120 further includes a fourth driving pump 1214, the third driving pump 1213 is used for pumping the lysis turbid liquid to the collecting device 115, and the fourth driving pump 1214 is used for pumping the neutralized liquid to the collecting device 115. It will be appreciated that the collection means 115 and the fourth drive pump 1214 are arranged to mix the lysis suspension and the neutralising solution in a predetermined ratio to enhance the neutralising effect of the neutralising solution on the lysis suspension.
The first driving pump 1211, the second driving pump 1212, the third driving pump 1213 and the fourth driving pump 1214 have various structures, and in an embodiment, the first driving pump 1211, the second driving pump 1212, the third driving pump 1213 and the fourth driving pump 1214 are membrane pumps. The diaphragm pump is a novel conveying machine, and has the advantages of being capable of conveying various corrosive liquids, liquids with particles, high-viscosity, volatile, inflammable and highly toxic liquids, and the first driving pump 1211, the second driving pump 1212, the third driving pump 1213 and the fourth driving pump 1214 can be made of plastic, aluminum alloy, cast iron or stainless steel materials.
Referring to fig. 2, in an embodiment, the plasmid vector continuous lysis apparatus 100 further includes a first connection pipe 131, a second connection pipe 132, a third connection pipe 133 and a fourth connection pipe 134, the first connection pipe 131 connects the first container 111 and the fourth container 114, the second connection pipe 132 connects the second container 112 and the first connection pipe 131, the third connection pipe 133 connects the fourth container 114 and the collection device 115, and the fourth connection pipe 134 connects the third container 113 and the third connection pipe 133. In this embodiment, the continuous plasmid vector lysis apparatus 100 further comprises a first tube connector 141 and a second tube connector 142; the first pipe joint 141 is arranged on the first connecting pipe 131, and the first pipe joint 141 comprises three joints, two joints are communicated with the first connecting pipe 131, and the third joint is communicated with one end of the second connecting pipe 132 away from the second container 112; the second pipe joint 142 is disposed on the third connecting pipe 133, and the second pipe joint 142 also includes three joints, two joints are communicated with the third connecting pipe 133, and the third joint is communicated with one end of the fourth connecting pipe 134 away from the third container 113. The first pipe joint 141 enables the re-suspended turbid liquid and the lysis solution to start to be mixed with each other at the first pipe joint 141, and the second pipe joint 142 enables the lysis turbid liquid and the neutralization solution to start to be mixed with each other at the second pipe joint 142, so that the continuity of the thallus lysis process is further ensured. The first pipe connector 141 and the second pipe connector 142 may also be respectively provided with a first valve and a second valve for regulating the flow rate of the turbid liquid flowing through the first pipe connector 141 and the second pipe connector 142.
In one embodiment, the continuous plasmid vector lysis apparatus 100 further comprises a resuspension solution, a lysis solution, and a neutralization solution. The components of the resuspension solution, the lysis solution and the neutralization solution belong to the prior art, and in all the embodiments, thalli can be continuously lysed by using the resuspension solution, the lysis solution and the neutralization solution in the prior art. However, the lysis effect of the resuspension, the lysis solution and the neutralization solution on the bacterial cells in the prior art is still not ideal, and the inventors have improved the lysis effect. The improved resuspension comprises Tris (hydroxymethyl) aminomethane (Tris) -hydrochloric Acid (HCl), Ethylene Diamine Tetraacetic Acid (EDTA) and glucose, wherein the concentration of the Tris (hydroxymethyl) aminomethane-hydrochloric Acid is 50mmol/L, the concentration of the Ethylene Diamine tetraacetic Acid is 10mmol/L, and the concentration of the glucose is 100mmol/L, wherein the Tris (hydroxymethyl) aminomethane-hydrochloric Acid mainly controls the pH value of the solution, and the Ethylene Diamine tetraacetic Acid mainly inhibits Deoxyribonuclease (DNase) to protect plasmids from enzymolysis, inhibit microbial growth, chelate divalent metal ions and the like; the improved lysis solution comprises Sodium hydroxide (NaOH) and Sodium Dodecyl Sulfate (SDS), the concentration of the Sodium hydroxide is 300mmol/L, the ratio of the weight to the volume (W/V) of the Sodium Dodecyl Sulfate is 1%, and the Sodium hydroxide is a main lysis component; the improved neutralization solution comprises potassium acetate (CH3COOK), the concentration of the potassium acetate is 3mol/L, K + decomposed by the potassium acetate can react with ethylene diamine tetraacetic acid in the neutralization process to generate water-insoluble potassium dodecyl sulfate, and the potassium dodecyl sulfate and a large amount of protein, host DNA and the like generate precipitates together to achieve the purpose of removing the potassium dodecyl sulfate.
The different volume ratios of the heavy suspension, the lysate and the neutralizing solution added for reaction have different effects on the thallus lysis. Referring to tables 1 to 4 and 3 to 5, in one embodiment, the inventors used the modified resuspension solution, lysis solution and neutralization solution in different volume ratios to compare the difference between the different volume ratios of the above solutions and the lysis effect of the bacteria.
In this embodiment, the conventional manual lysis method is performed as follows:
1. weighing about 50g of thallus;
2. re-suspending the thalli by using the heavy suspension, and pouring the re-suspended turbid liquid into a 5L blue-cap bottle when the thalli are in a non-blocky state;
3. adding the lysis solution into the blue-cap bottle, and continuously and gently shaking the blue-cap bottle to crack the thalli, wherein the cracking time is not more than 5 min;
4. rapidly adding the neutralizing solution into the cracking turbid solution, and stopping neutralizing after the neutralizing turbid solution completely forms a precipitate;
5. centrifuging the neutralized turbid solution at 6000g and 4 deg.C for 30min, and collecting supernatant;
6. filtering the supernatant with a Sachs filter with the diameter of a filter hole of 1 mu m, and collecting filtrate;
7. determining and comparing the amount of HCP remaining in the permeate;
8. determining and comparing the residual amount of HCD in the filtrate;
9. and concentrating the filtrate to form a concentrated solution, calculating and comparing the supercoiled proportion in the concentrated solution, wherein the content of RNA is not counted during calculation.
In the above steps, the volumes of the resuspension solution, the lysis solution and the neutralization solution added in the reaction are shown in table 1, and the volume ratio of the resuspension solution, the lysis solution and the neutralization solution is 1: 1: the experimental group of 1 was named first group, and the volume ratio was 2: 4: the experimental group of 3 was named as a second group, and the first and second groups included two experiments, respectively. The HCP content determination and analysis results of the filtrates of the first and second groups are shown in table 2 and fig. 3; the results of the determination and analysis of the HCD content of the permeate of the first and second groups are shown in table 3 and fig. 4, and the results of the calculation and analysis of the supercoiled proportion of the concentrate of the first and second groups are shown in table 4 and fig. 5. HCP, HCD content determination and analysis method and supercoiled proportion calculation and analysis method belong to the prior art, and are not described herein.
Referring to Table 2 and FIG. 3, the mean HCP content of the first group was (3.135. + -. 0.545). times.108The average HCP content of the filtrates of the second group was (2.27. + -. 0.11). times.108The mean value of the HCP of the second group was less than the mean value of the first group (P0.2600), indicating that the second group precipitated HCP impurities better than the first group. Referring to Table 3 and FIG. 4, the average HCD content of the first group of the filtrates was (1.91. + -. 0.21). times.106The average value of the HCD content of the filtrate of the second group is (1.74 +/-0.075) multiplied by 106(iii) the mean value of the permeate HCD of the second group is less than the mean value of the first group (P ═ 0.536)4) Indicating that the second group precipitated HCP impurities better than the first group. Referring to table 4 and fig. 5, the mean value of the supercoiled proportions of the concentrates of the first group is 76.35 ± 8.25, the mean value of the supercoiled proportions of the concentrates of the second group is 84.15 ± 5.85, and the mean value of the supercoiled proportions of the concentrates of the second group is greater than the mean value of the first group (P ═ 0.5212), which indicates that the purity of the plasmids extracted by the second group is higher than that of the first group. Combining the above data, the volume ratio was 2: 4: 3, the lysis effect of the resuspension, the lysate and the neutralization solution on the thalli is better than that of a mixture of the resuspension, the lysate and the neutralization solution with a volume ratio of 1: 1: 1 cracking effect.
The following are the details of tables 1 to 4.
TABLE 1 volumes and volume ratios of the resuspension, the lysis solution, and the neutralization solution added to the reaction
| Group number | Volume ratio | Resuspension (ml) | Lysis solution (ml) | Neutralizing solution (ml) |
| First group | 1：1：1 | 500 | 500 | 500 |
| Second group | 2：4：3 | 250 | 500 | 375 |
TABLE 2 results of HCP content determination of the filtrates from the first and second set of experiments
TABLE 3 results of HCD content measurement of the filtrates from the first and second sets of experiments
TABLE 4 results of calculation of supercoiled proportion of the concentrates of the first and second set of experiments
Referring to tables 5-7, and fig. 6-8, in one embodiment, the investigator uses a modified volume ratio of 2: 4: 3, comparing the effect of the plasmid vector continuous lysis device 100 on the lysis of the bacteria by the traditional manual lysis method. For the operation steps of lysing bacteria by the conventional manual lysis method, please refer to the previous embodiment, which is not described herein again. The following describes the procedure of the plasmid vector continuous lysis apparatus 100 for lysing bacterial cells.
Referring to FIG. 3, in this embodiment, the operation steps of the plasmid vector continuous lysis apparatus 100 for lysing bacteria are as follows:
1. resuspending 50g of thalli by 250ml of the heavy suspension, pouring the thalli into the first container 111 after the thalli are in a blocky state, adding 500ml of the lysis solution into the second container 112, and adding 500ml of the neutralization solution into the third container 113;
2. simultaneously turning on the first driving pump 1211 and the third driving pump 1213, wherein the flow rate of the re-suspended turbid solution is set to 50ml/min, and the flow rate of the lysis solution is set to 100 ml/min;
3. under the driving of the first driving pump 1211 and the second driving pump 1212, the resuspended turbid solution and the lysate respectively flow into the fourth container 114, and the resuspended turbid solution and the lysate are immediately timed when entering the fourth container 114;
4. after timing for 5min, simultaneously starting the third driving pump 1213 and the fourth driving pump 1214, wherein the flow rate of the lysate turbid liquid is set to be 150ml/min, and the flow rate of the neutralization liquid is set to be 75 ml/min;
5. the lysis suspension in the third container 113 and the neutralization solution in the fourth container 114 enter the collection device 115 under the driving of the third driving pump 1213 and the fourth driving pump 1214;
6. centrifuging the neutralized turbid liquid in the collection device 115 for 30min at 6000g and 4 ℃, and taking supernatant;
7. filtering the supernatant with a Sachs filter with the diameter of a filter hole of 1 mu m, and collecting filtrate;
8. determining and comparing the amount of HCP remaining in the permeate;
9. determining and comparing the residual amount of HCD in the filtrate;
10. and concentrating the filtrate to form a concentrated solution, calculating and comparing the supercoiled proportion in the concentrated solution, wherein the content of RNA is not counted during calculation.
Referring to Table 5 and FIG. 6, the HCP content of the permeate of the conventional manual lysis method was (2.975. + -. 0.135). times.10 on average5The average value of the HCP content of the filtrate of the continuous lysis device 100 is (2.965. + -. 0.335). times.108The mean value of the HCP of the permeate of the continuous lysis device 100 was less than the mean value of the conventional manual lysis method (P-0.9804), indicating the precipitation effect of the continuous lysis device 100 on HCP impuritiesBetter than the conventional manual lysis method. Referring to Table 6 and FIG. 7, the average HCD content of the filtrate of the conventional manual cracking method was (1.063. + -. 0.298). times.103The average HCD content of the filtrate of the continuous pyrolysis apparatus 100 is (0.568. + -. 0.113). times.103The average value of the filtrate HCD of the continuous cracking apparatus 100 is smaller than the average value of the conventional manual cracking method (P ═ 0.2559), indicating that the continuous cracking apparatus 100 has a better precipitation effect on HCD impurities than the conventional manual cracking method. Referring to table 7 and fig. 8, the average value of the supercoiled proportion of the concentrated solution of the conventional manual lysis method is 76.55 ± 3.65, the average value of the supercoiled proportion of the concentrated solution of the continuous lysis device 100 is 84.45 ± 1.15, and the average value of the supercoiled proportion of the concentrated solution of the continuous lysis device 100 is greater than the average value of the conventional manual lysis method (P ═ 0.2631), which indicates that the purity of the plasmid extracted by the continuous lysis device 100 is higher than that of the conventional manual lysis method. In combination with the above data, the plasmid vector continuous lysis device 100 has a better lysis effect on the bacteria than the traditional manual lysis method.
The following are the details of tables 5 to 7.
TABLE 5 results of HCP content measurement of permeate from conventional hand-operated lysis and continuous lysis apparatus 100
TABLE 6 detection results of HCD content of filtrate of the conventional manual cracking and continuous cracking apparatus 100
TABLE 7 calculation results of supercoiled proportion of concentrate of the conventional manual cracking and continuous cracking apparatus 100
In order to achieve a better lysis effect, the flow rate ratio of the resuspended turbid solution, the lysate turbid solution and the neutralizing solution is better consistent with the volume ratio of the resuspended turbid solution, the lysate turbid solution and the neutralizing solution, in this embodiment, the volume ratio of the resuspended turbid solution, the lysate turbid solution and the neutralizing solution is 2: 4: 6: 3, whereby the flow rate ratio of the resuspended suspension, the lysate suspension and the neutralizer is also 2: 4: 6: 3. of course, when the volumes and volume ratios of the resuspension solution, the lysate and the neutralizing solution added into the plasmid vector continuous lysis device 100 are different, the flow rates and flow rate ratios of the resuspension turbid solution, the lysate turbid solution and the neutralizing solution are also adjusted accordingly. For example, in another embodiment, the volume ratio of the resuspension, the lysis solution, and the neutralization solution is 1: 1: 1, the ratio of the flow rates of the resuspended turbid solution, the lysate turbid solution, and the neutralizer is also 1: 1: 2: 1.
in order to mix the resuspended turbid solution and the lysis solution quickly and uniformly, so as to achieve a better bacteria lysis effect, please refer to fig. 2, in an embodiment, the plasmid vector continuous lysis apparatus 100 further includes a stirring member 150, and the stirring member 150 is disposed in the fourth container 114 and is used for stirring the lysed turbid solution in the fourth container 114. The stirring member 150 has various structures, for example, in this embodiment, the stirring member 150 includes a magnetic rotor 151 and a driving seat 152, the magnetic rotor 151 is disposed at the bottom end of the inside of the fourth container 114, the driving seat 152 is correspondingly disposed at the outside of the bottom end of the fourth container 114, and the driving seat 152 is used for driving the magnetic rotor 151 to rotate in the fourth container 114. In order to accelerate the neutralization of the lysis suspension by the neutralizing solution, the stirring element 150 can also be arranged in the collection device 115.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (10)
1. A continuous lysis device for a plasmid carrier, comprising:
the reaction container comprises a first container, a second container, a third container and a fourth container, wherein the first container is used for containing a heavy suspension turbid liquid comprising thalli and a heavy suspension liquid, the second container is used for containing a lysis solution, the third container is used for containing a neutralization solution, and the fourth container is used for containing a lysis turbid liquid comprising the heavy suspension turbid liquid and the lysis solution; and
the driving assembly comprises a first driving pump, a second driving pump and a third driving pump, the first driving pump is used for pumping the heavy suspension turbid liquid in the first container into the fourth container, the second driving pump is used for pumping the lysis liquid in the second container into the fourth container, and the third driving pump is used for pumping the lysis turbid liquid in the fourth container into the third container.
2. The continuous plasmid vector lysis device of claim 1, wherein said reaction vessel further comprises a collection device for holding a neutralized turbid solution comprising said lysed turbid solution and said neutralized solution, said drive assembly further comprises a fourth drive pump for pumping said lysed turbid solution into said collection device, said fourth drive pump for pumping said neutralized solution into said collection device.
3. The continuous plasmid vector lysis device according to claim 2, wherein said continuous plasmid vector lysis device further comprises a first connection tube, a second connection tube, a third connection tube and a fourth connection tube, said first connection tube connects said first container and said fourth container, said second connection tube connects said second container and said first connection tube, said third connection tube connects said fourth container and said collection device, and said fourth connection tube connects said third container and said third connection tube.
4. The continuous lysis device of claim 1, wherein said first actuation pump, said second actuation pump and said third actuation pump are diaphragm pumps.
5. The continuous lysis device for plasmid vector according to any one of claims 1 to 4, wherein said continuous lysis device for plasmid vector further comprises a resuspension solution, a lysis solution and a neutralization solution, wherein the volume ratio of said resuspension solution to said lysis solution to said neutralization solution is 2: 4: 3.
6. the continuous plasmid vector lysis device according to claim 5, wherein said resuspension solution comprises tris (hydroxymethyl) aminomethane-hydrochloric acid, ethylenediaminetetraacetic acid, and glucose, wherein the concentration of said tris (hydroxymethyl) aminomethane-hydrochloric acid is 50mmol/L, the concentration of said ethylenediaminetetraacetic acid is 10mmol/L, and the concentration of said glucose is 100 mmol/L.
7. The continuous lysis device of plasmid vector according to claim 5, wherein the lysis solution comprises sodium hydroxide and sodium dodecyl sulfate, the concentration of the sodium hydroxide is 300mmol/L, and the ratio of the weight to the volume of the sodium dodecyl sulfate is 1%.
8. The continuous lysis apparatus for plasmid vector according to claim 5, wherein the neutralization solution comprises potassium acetate, and the concentration of the potassium acetate is 3 mol/L.
9. The continuous lysis apparatus of any one of claims 1 to 4, wherein said reaction vessel is a reaction barrel, a reaction tank, a reaction flask or a reaction tank.
10. The continuous lysis device of any one of claims 1 to 4, wherein said continuous lysis device further comprises a stirring member disposed in said fourth container for stirring said lysis suspension in said fourth container.
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| CN202010908456.3A CN111979109A (en) | 2020-09-01 | 2020-09-01 | Plasmid vector continuous cracking device |
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