JP2000210070A - Centrifugal separation vessel and liquid layer moving method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a centrifugal separation vessel capable of safely, easily and surely recovering supernatant from a microplate hole by using a specific microplate of a 1st vessel and a specific 2nd vessel capable of holding the liquid phase. SOLUTION: This centrifugal separation vessel holding a solid phase and a liquid phase in plural holes and capable of safely, easily and surely recovering a supernatant from a microplate hole is produced by providing a 1st vessel composed of a microplate 3 and a 2nd vessel 4 capable of holding the liquid phase 6 in the 1st vessel, placing the 1st and the 2nd vessels in a swingable basket 2 rotating around a rotary axis 1 in a manner to satisfy the formula F1>F2 wherein F1 is the force to hold the above solid phase 5 in the case of directing the opening of the 1st vessel in the direction of gravity and F2 is the force necessary for moving the liquid phase out of the 1st vessel, and constituting the 2nd vessel to have a space larger than the total volume of the liquid existing in the plural holes of the 1st vessel.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a centrifugal separation vessel for separating a solid phase and a liquid phase in a microplate, and a liquid layer moving method using the same.

[0002]

2. Description of the Related Art A microplate is provided with a plurality of microcapacity holes (about 0.2 to 2 ml), and is a substitute for culturing bacteria. For example, when extracting plasmid DNA from Escherichia coli, the culture solution of Escherichia coli containing the plasmid DNA is centrifuged to collect the bacteria, unnecessary supernatant is removed, and then a bacterial suspension is added to the solution. The bacteria are uniformly present, and a lysis reagent is added to lyse the bacteria and simultaneously denature unnecessary genomic DNA. Next, a neutralizing reagent is added to neutralize the solution, the bacterial cell residue is removed as a precipitate by centrifugation, and the supernatant containing the plasmid DNA is transferred to a clean microplate well. Then, the supernatant containing the plasmid DNA is mixed with isopropyl alcohol to precipitate the plasmid DNA. The precipitated plasmid DNA was recovered by centrifugation, and unnecessary supernatant was removed. Then, 70% ethanol was added to the plasmid DNA precipitated by centrifugation, and the plasmid DNA was removed.
Is washed, and the plasmid DNA is collected by centrifugation to remove unnecessary supernatant.

[0003] "Isolation of plasmid DNA by an automated apparatus" will be described below in detail with reference to FIG.

A 1-well 2 ml 96-well microplate
A 0.3 ml well of 2YT medium was injected, and the plasmid p
E. coli JM101 transformed with UC19 was inoculated with 37
Incubate at ℃ overnight. After the cultivation, the microplate is set on the alignment unit, and parameters such as the number of samples, centrifugation conditions, and addition amounts of various reagents are input to the control unit, and the operation is started by pressing the start button. (1) Bacterial Collection Step The microplate is accommodated at a predetermined position of a rotor in a centrifuge by a transfer device including a hand mechanism. When a predetermined number of microplates are accommodated, 5,700 rpm (5,010 ×
g), centrifugation for 7 minutes is started to separate the cells containing the plasmid DNA and the culture solution. Thereafter, the microplate is transferred to the decanting unit by the transfer device,
Subsequently, unnecessary supernatant is removed by suction using an automatic pipetting device. (2) Lysis Step The microplate containing the cells was transferred to a reagent injection part, and a 0.02 ml cell suspension (50 mM glucose, 1 mM glucose,
0 mM EDTA, 10 mM Tris-HCl, pH7.
5) is injected. Next, the microplate is transferred to a suspension part to suspend the bacteria uniformly in the solution. Thereafter, the microplate is transferred to the reagent injection part, and 0.04 ml of the microplate is transferred to the reagent injection part.
Lysis reagent (1% SDS, 0.2N NaOH) is injected. Next, the microplate was transferred to the suspension part, and both liquids were sufficiently suspended. Thereafter, the microplate was transferred to the reagent injection part, and 0.03 ml of the neutralizing reagent (3M CH 3
3COOK, 5M CH3COOH) is injected. Next, the microplate is transferred to a suspension part, and both liquids are sufficiently suspended. Then, when the microplate is accommodated in a predetermined position of the rotor in the centrifuge by the transfer device, 5,700 rpm (5,010 ×
g), centrifugation for 15 minutes is started and plasmid DN
A is separated into a supernatant containing A and a precipitate consisting of cell residue.
Thereafter, the microplate is transferred to the supernatant transfer section by the transfer device, and the supernatant is transferred to a new microplate by an automatic pipetting device equipped with a disposable pipette tip. Then, the microplate containing the bacterial cell residue is discarded at a predetermined place by the transfer device. (3) Plasmid DNA recovery step The new microplate is transferred to the reagent injection section by the transfer device, and 0.1 ml of a DNA recovery reagent (isopropyl alcohol) is injected. Next, the microplate was moved to the suspension section by a moving device, and both solutions were sufficiently suspended.
The plasmid DNA is precipitated. Then, when the microplate is accommodated in a predetermined position of the rotor in the centrifuge by the transfer device, 5,700 rpm is given by a command from the control unit.
Centrifugation at pm (5,010 × g) for 20 minutes is started to separate the plasmid DNA into a precipitate and isopropyl alcohol. Thereafter, the microplate is transferred to a decanting unit by a transfer device, and unnecessary supernatant is suctioned and removed by an automatic pipetting device. (4) Plasmid DNA Washing Step The new microplate is transferred to the reagent injection section by the transfer device, and 0.3 ml of the DNA washing reagent (70% ethanol) is injected. Next, the microplate is transferred to a suspension part, and both liquids are sufficiently suspended. Then, when the microplate is accommodated in a predetermined position of the rotor in the centrifuge by the transfer device, 5,70 in accordance with a command from the control unit.
Centrifugation at 0 rpm (5,010 × g) for 20 minutes is started, and the DNA is separated into a precipitate of plasmid DNA and ethanol. Thereafter, the microplate is transferred to a decanting unit by a transfer device, and unnecessary supernatant is suctioned and removed by an automatic pipetting device.

[0005] Therefore, the plasmid DNA obtained as a precipitate through each of the above steps (1) to (4) is dissolved in sterilized distilled water or the like and used for a sequencing reaction or the like.

[0006] In such a plasmid DNA extraction method, there are cases where the supernatant is unnecessary (when removing the supernatant) and cases where the supernatant is needed (when the supernatant is collected). Describe.

[0007] In the above plasmid DNA extraction method, (1) in the cell collection step, after centrifugation, the cells containing the plasmid DNA are separated into a culture solution. in this case,
The supernatant is removed because the culture solution that is the supernatant is unnecessary. Such removal of the supernatant is carried out in the step (3) in the step of recovering the plasmid DNA, the precipitation of the plasmid DNA after centrifugation and the isopropanol to isopropanol.
(4) In the washing step of the plasmid DNA, the plasmid DNA after centrifugation is precipitated and 70% ethanol is applied to 70% ethanol. Further, in the (2) lysis step, centrifugation is performed to separate a precipitate consisting of cell residues and a supernatant containing plasmid DNA. In this case, the supernatant contains plasmid DNA, and the supernatant is necessary and must be collected. Thus, in the experimental operation, when the supernatant after centrifugation is unnecessary (when the supernatant is removed)
(When collecting the supernatant). Hereinafter, a method of removing or collecting a supernatant from a microplate in a conventional general experimental operation will be described.

First, when removing the supernatant, the opening of the microplate is oriented in the direction of gravity to allow the solution in the microplate hole to flow out naturally. However, when the solution does not flow out naturally due to the surface tension of the supernatant, the microplate is vigorously shaken. The supernatant is forcibly discharged by shaking. In addition to the above-mentioned method, there is a method of aspirating and removing unnecessary supernatant from the inside of a microplate using a manual pipette or an automatic machine having a pipette function.

Conversely, when collecting the supernatant, the necessary supernatant is transferred from the microplate hole to another microplate hole using a manual pipette or an automatic machine equipped with a pipette function from each microplate hole using a disposable pipette tip. The transition has been made in the way. In addition,
Unlike the case where unnecessary supernatant is removed, it is very difficult to transfer the supernatant to each well of another microplate one by one by natural flow.
No such approach has been taken.

[0010]

The problems that occur when removing the supernatant from the microplate hole or when recovering the supernatant will be described below.

First, a problem in removing unnecessary supernatant from the microplate will be described. As a method of holding a required solid layer in a microplate and forcibly discharging unnecessary supernatant, there is a method of vibrating the microplate vigorously. In the case of such a method, there is a possibility that the supernatant contaminated by the bacteria is scattered around and the supernatant is mixed into the holes from the holes of the microplate. In this case, contamination occurs in which bacteria existing only in a specific hole of the microplate are mixed into another hole. In addition, if the scattering of the bacteria reaches surrounding laboratory instruments and the fingers of the experimenter, it adversely affects other samples performed using the laboratory instruments and the human body of the experimenter using them. There was a problem. In addition, as a method that does not cause the above-described problem, there is a method of removing unnecessary supernatant using a manual pipette.In this case, since a small amount of supernatant must be removed by suction from each hole of the microplate, There is a problem that the operation is very troublesome and troublesome, and it is not possible to improve the operation efficiency. On the other hand, work efficiency can be improved by using an automatic machine having a pipette function. However, since this automatic machine is an expensive device, there is a problem that the cost increases.

Next, problems in collecting the necessary supernatant in the microplate will be described. At the time of collection, use a micropipette that can suck and discharge a small amount of liquid,
At the time of aspiration and discharge of the supernatant, a disposable tip is attached to the part that comes into contact with the liquid, and the supernatant is aspirated with a micropipette. There was a problem that it was not possible. In addition, when collecting the necessary supernatant using a manual pipette, the supernatant must be collected from each hole of the microplate, which is extremely troublesome and time-consuming, resulting in an increase in work efficiency. There was a problem that improvement could not be achieved. On the other hand, work efficiency can be improved by using an automatic machine having a pipette function. However, since this automatic machine is an expensive device, there is a problem that the cost increases.

An object of the present invention is to provide an inexpensive centrifugal separation container capable of solving the above-mentioned problems and reliably collecting and removing a supernatant from a microplate hole by a safe and simple method, and a method of moving a liquid layer thereof. It is to be.

[0014]

SUMMARY OF THE INVENTION The object of the present invention is to dispose unnecessary openings in the microplate holes so that the openings of the microplate holes are oriented in the direction of gravity. This is achieved by collecting the supernatant that has not flowed out into a container having a volume equal to or greater than the total volume of the supernatant in the wells of the microplate. Here, the force F1 for causing sedimentation in the microplate hole and the force F2 for moving the liquid phase out of the container as the force for holding the solid phase are F
1> F2.

Further, a force for moving the liquid phase to a first container comprising a microplate and a second container capable of accommodating the liquid phase in the first container and, if necessary, being divided corresponding to each microplate hole. The relationship between F2 and the force F1 for holding the solid phase in the microplate is achieved by satisfying the relationship of F1> F2.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a method for separating a solid phase and a liquid phase in this embodiment will be described.

FIG. 1 shows each state of the sediment 5 and the supernatant 6 when the swing rotor having the swingable bucket 2 for accommodating the microplate 3 and the supernatant collecting container 4 is rotated around the rotation axis 1. (A) is a cross-sectional view showing a state in which the supernatant in the hole does not flow out naturally due to surface tension or the like when the microplate hole is arranged in the bucket 2 with the hole facing in the direction of gravity, and (b) is a bucket. FIG. 3 is a cross-sectional view showing the state of the precipitate 5 and the supernatant 6 during centrifugation in which the rocker 2 rocks;
(C) is a cross-sectional view showing the state of the precipitate 5 and the supernatant 6 after centrifugation, and FIG. 2 is a perspective view showing the microplate 3 and the supernatant collection container 4.

Hereinafter, "isolation of plasmid DNA by an automated apparatus" will be described in detail with reference to FIG.

One 96-well microplate for 2 ml per well
A 0.3 ml well of 2YT medium was injected, and plasmid pUC was added.
After transformation with E. coli 19, E. coli JM101 was inoculated and 37
Incubate at ℃ overnight. After the cultivation, the microplate is set on the alignment unit, and parameters such as the number of samples, centrifugation conditions, and addition amounts of various reagents are input to the control unit, and the operation is started by pressing the start button. (1) Bacterial Collection Step The microplate is accommodated at a predetermined position of a rotor in a centrifuge by a transfer device including a hand mechanism. When a predetermined number of microplates are accommodated, 5,700 rpm (5,010 ×
g) Centrifugation for 7 minutes is started and plasmid DNA
And a culture solution containing the cells. The microplate is moved to the standby unit by the transfer device, the microplate is inverted by the reversing device in the standby unit, and then the container for discarding the supernatant is accommodated in the rotor by the transfer device, and the inverted microplate is discarded for the supernatant. Place on container. According to a command from the control unit, centrifugation is performed at 360 rpm (20 × g) for 1 second, and unnecessary supernatant in the microplate is transferred to a waste container. The microplate is transferred by the transfer device.
The waste container is transferred to the standby unit, inverted by the inverting device, and transferred to the predetermined place by the transfer device. (2) Lysis Step The microplate containing the cells was transferred to the reagent injection section by a transfer device, and 0.02 ml of the bacterial suspension (50 mM glucose, 10 mM EDTA, 10 mM Tris-HCl,
pH 7.5) is injected. Next, the microplate is transferred to the suspension part by the transfer device to suspend the bacteria uniformly in the solution. Thereafter, the microplate is transferred to the reagent injection part by the transfer device, and 0.04 ml of the lysis reagent (1) is transferred. % SDS, 0.2N NaOH) is injected. Next, the microplate is transferred to the suspension part, and both liquids are sufficiently suspended. Thereafter, the microplate is transferred to the reagent injection part, and 0.03 ml of the neutralizing reagent (3M CH3COO) is transferred.
K, 5M CH3COOH) is injected. Next, the microplate is transferred to a suspension part and both liquids are sufficiently suspended. Then, when the microplate is accommodated in a predetermined position of the rotor in the centrifuge by the transfer device, 5,700 rpm (5,010 × g), 1,
Centrifugation for 5 minutes is started, and the supernatant is separated into a supernatant containing plasmid DNA and a precipitate consisting of bacterial cell residues. afterwards,
The microplate is transferred to the standby unit by the transfer device, the microplate is inverted by the reversing device in the standby unit, and the new microplate is stored in the rotor by the transfer device, and the inverted microplate is transferred to the new microplate by the transfer device. Place on plate. Then, according to a command from the control unit, 360 rpm (20 × g), 1
After the centrifugation for 2 seconds is started and the supernatant in the microplate is transferred to a new microplate, the microplate containing the bacterial cell residue is discarded in a predetermined place by the transfer device. (3) Plasmid DNA recovery step The new microplate is transferred to the reagent injection section by the transfer device, and 0.1 ml of a DNA recovery reagent (isopropyl alcohol) is injected. Next, the microplate was transferred to the suspension section by a transfer device, and both solutions were sufficiently suspended.
The plasmid DNA is precipitated. Then, when the microplate is accommodated in a predetermined position of the rotor in the centrifuge by the transfer device, 5,700 rpm is given by a command from the control unit.
Centrifugation at pm (5,010 × g) for 20 minutes is started to separate the plasmid DNA into a precipitate and isopropyl alcohol. Thereafter, the microplate is transferred to the standby unit by the transfer device, the microplate is inverted by the reversing device in the standby unit, and then the container for discarding the supernatant is stored in the rotor by the transfer device, and then the inverted microplate is transferred. Place on the container for supernatant disposal by the device.
Next, according to a command from the control unit, 360 rpm (20 ×
g), centrifugation for 1 second is started, and unnecessary supernatant in the microplate is transferred to a disposal container. Then, the microplate is transferred to a standby unit, inverted by an inverting device, and inverted by a transfer device. Is transported to a predetermined place. (4) Plasmid DNA Washing Step The new microplate is transferred to the reagent injection section by the transfer device, and 0.3 ml of the DNA washing reagent (70% ethanol) is injected. Next, the microplate is transferred to a suspending section to suspend both solutions. When the microplate is accommodated in a predetermined position of the rotor in the centrifuge by the transfer device, 5,700 rpm is instructed by the control unit.
(5,010 × g), centrifugation for 20 minutes is started,
It is separated into the plasmid DNA precipitate and ethanol.
Thereafter, the microplate is transferred to the standby unit by the transfer device, the microplate is inverted by the reversing device in the standby unit, and then the container for discarding the supernatant is stored in the rotor by the transfer device, and then the inverted microplate is transferred. Place on the container for supernatant disposal by the device. Next, centrifugation at 360 rpm (20 × g) for 1 second is started by a command from the control unit, and unnecessary supernatant in the microplate is transferred to a waste container, and then the microplate is transferred to the standby unit by the transfer device. Then, the container is turned over by the reversing device and the container for disposal is transferred to a predetermined place by the transfer device.

Therefore, the plasmid DNA obtained as a precipitate through each of the above steps (1) to (4) is dissolved in sterilized distilled water or the like and used for a sequencing reaction or the like.

The microplate and the waste container used in such a plasmid DNA extraction method are configured as shown in FIGS. 1 and 2, and are placed in the bucket 2 as shown in FIG. Is provided with a supernatant collection container 4, and a microplate 3 is further provided on the supernatant collection container 4. At this time, the openings of the plurality of holes provided in the microplate 3 are arranged so as to face the direction of gravity (downward in FIG. 1). Note that a precipitate 5 and a supernatant 6 are provided in each hole. When such a bucket 2 is mounted on a swing rotor and the swing rotor is rotated by the rotating shaft 1 by the rotational power of a motor (not shown), the bucket 2 swings as shown in FIG. Only the supernatant 6 arranged in each hole moves outward (the side opposite to the drive shaft 1) by centrifugal force and accumulates in the supernatant collection container 4. Then, when the rotation of the swing rotor is stopped, the swing rotor returns to the state shown in FIG. Thereby, the supernatant 6 can be collected in the supernatant collection container 4. Here, by configuring the microplate 3 and the solution collection container 4 as shown in FIG. 2, the microplate 3 and the solution collection container 4 come into close contact with each other due to the centrifugal force generated during centrifugation. It is possible to prevent the supernatant 6 from scattering outside. here,
As shown in FIG. 7, a packing 7 made of a flexible material such as rubber is preferably provided between the microplate 3 and the solution recovery container 4 to further enhance the adhesion.

As described above, in the present embodiment, the microplate 3 is formed by a simple method using a centrifuge.
The supernatant 6 can be easily and safely collected and removed from the holes.

FIG. 3 is a perspective view and a sectional view showing another embodiment of the microplate 3 and the solution recovery container 4 shown in FIG. 1. By adopting such a configuration, the microplate shown in FIG. The supernatant 6 can be collected on a one-to-one basis in the solution collection containers 4 respectively corresponding to the respective holes of No. 3.

In the plasmid DNA extraction protocol shown in FIG. 5, in the lysis step, a plurality of necessary reagents are supplied to the microplate holes in a one-to-one correspondence, and the reaction proceeds. After centrifugation, the supernatant 6 containing the plasmid DNA is separated from the bacterial cell residue. However, when the supernatant is directed in the direction of gravity of the opening of the container depending on the amount of the supernatant, the supernatant 6 may flow out spontaneously. In such a case, the structure shown in FIG. 4 can prevent the supernatant from flowing out naturally even when the opening of the container is directed in the direction of gravity.

In FIGS. 3 and 4, when collecting the necessary supernatant in the microplate hole, a partition corresponding to the microplate hole is provided in the container so that the supernatant can be collected one by one from the microplate hole. Can be recovered. Here, if the necessary supernatant spontaneously flows out by orienting the opening of the microplate hole in the direction of gravity, it is necessary to provide a small hole in each hole of the microplate to prevent the spontaneous flow of the supernatant. Alternatively, a small hole is provided in the packing sandwiched between the microplate and the container in a one-to-one correspondence with the hole. Also, the centrifugal force F2 for transferring the supernatant
Can be made seismic. Furthermore, by providing a partition corresponding to the microplate hole in the container and providing a small hole that prevents the collected solution from spontaneously flowing out even when the collected solution is directed to the hole opening in the direction of gravity, the reagent etc. When supplied in a microplate container, several kinds of reagents necessary for the lysis step of plasmid DNA extraction are supplied to the microplate holes in a one-to-one correspondence, thereby reacting and unnecessary precipitation after centrifugation. Only the supernatant from the necessary supernatant can be transferred to another microplate.

[0026]

According to the present invention, it is possible to provide an inexpensive centrifugal separation vessel capable of securely collecting and removing a supernatant from a microplate hole by a safe and simple method, and a method for moving a liquid layer thereof. .

[Brief description of the drawings]

FIG. 1 shows respective states of a precipitate and a supernatant when a swing rotor having a swingable bucket accommodating a microplate and a supernatant recovery container according to the present invention is rotated around a rotation axis, a) is a cross-sectional view showing a state in which the supernatant in the hole does not flow out naturally due to surface tension or the like when the microplate hole is arranged in the bucket with the hole facing in the direction of gravity; FIG. 3C is a cross-sectional view showing the state of the precipitate and the supernatant in the inside, and FIG. 4C is a cross-sectional view showing the state of the precipitate and the supernatant after the centrifugation.

FIG. 2 is an external perspective view showing a microplate and a supernatant collection container according to the present invention.

FIG. 3 shows another microplate and a supernatant collection container according to the present invention, and is a transmission diagram and a cross-sectional view of a supernatant collection container corresponding one-to-one to microplate holes.

FIG. 4 shows still another microplate and supernatant collection container according to the present invention, and is a transmission diagram and a cross-sectional view of a supernatant collection container corresponding one-to-one to microplate holes.

FIG. 5 is an explanatory diagram showing a separation procedure of plasmid DNA extraction according to the present invention.

FIG. 6 is an overall configuration diagram showing a plasmid DNA extraction device according to the present invention.

FIGS. 7A and 7B show a packing disposed between a microplate and a solution collection container according to the present invention, wherein FIG. 7A is a front view and FIG. 7B is a side view.

FIG. 8 is an overall configuration diagram showing a conventional plasmid DNA extraction device.

[Explanation of symbols]

Numeral 3 denotes a microplate serving as a first container, and numeral 4 denotes a second container.

Claims (5)

[Claims] 1. A centrifugal separation container in which a solid phase and a liquid phase are accommodated in a plurality of holes, wherein the centrifugal separation container is a first container comprising a microplate and the liquid phase in the first container. And a second container capable of containing the first container.
When the opening of the container is oriented in the direction of gravity, the first container and the first container may be configured such that the relationship between the force F1 for holding the solid phase and the force F2 for moving the liquid phase out of the first container is F1> F2. The centrifugal separator, wherein the second container is formed, and the second container is configured to have a space equal to or larger than the total volume of the liquid present in the plurality of holes formed in the first container. container. 2. The centrifugal separation container according to claim 1, wherein the force F2 is generated by a centrifugal force or a seismic force. 3. The centrifugal centrifuge according to claim 1, wherein the second container is divided corresponding to each microplate hole, and each divided part has a space larger than a liquid phase can be held. Separation container. 4. A small hole for preventing a retained solution phase from spontaneously flowing out when an opening provided in a divided portion of the second container is directed in the direction of gravity. The centrifugal separation container according to claim 3, characterized in that: 5. A method for moving a liquid layer in a first container into a second container in a centrifugal separation container containing a solid phase and a liquid phase in a plurality of holes, wherein the liquid phase The liquid layer moving method, wherein the force F2 for moving the liquid phase is F1> F2 with respect to the force F1 for holding the solid phase in the first container.