EP1899044A2

EP1899044A2 - Device for the rotor-stator homogenization of heterogeneous samples, and use of such a device

Info

Publication number: EP1899044A2
Application number: EP06762237A
Authority: EP
Inventors: Frank Vitzthum
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-07-04
Filing date: 2006-06-28
Publication date: 2008-03-19
Also published as: DE102005031459A1; US20100116744A1; WO2007003318A2; WO2007003318A3

Abstract

Disclosed is a rotor-stator homogenization device which is characterized in that a rotor-stator homogenizer (2) is connected to a reaction vessel (5a) via a joining system (8), and/or homogenization surfaces (7) are used, and/or a rotor-stator homogenizer is used that is suitable for transferring material. Also disclosed is a method which utilizes said device and is characterized in that the reproducibility, efficiency, yield, waste disposal, automation of the rotor-stator homogenization, etc. is improved, for example, while the risk of contaminations is reduced.

Description

Apparatus and method for rotor-stator homogenization

The present invention relates to a device for the homogenization of inhomogeneous samples, in particular for the homogenization or disintegration of immiscible or difficult to mix liquids or solid materials.

As a rule, rotor-stator homogenizers consist of an outer shaft tube, the stator, and a rotatably mounted rotor shaft located therein. This rotor shaft is generally driven via a clutch by means of a motor. The homogenization of samples takes place via correspondingly shaped comminution regions of the rotor and of the stator during the rotational movement of the rotor. In general, a rotor-stator homogenizer is manually introduced into a reaction vessel or a homogenization vessel, in which the sample and optionally a digestion solution is located. Here, the position of the rotor-stator homogenizer in the vessel is not defined, so that the reproducibility of Homogenisationsprozessen is limited.

The samples to be homogenized are, for example, suspensions and emulsions of immiscible liquids. The homogenization of such suspensions and emulsions is of interest in many fields, such as in the manufacture of foods, cosmetics, pharmaceuticals, paints, etc. Rotor-stator homogenizers are also used for the homogenization of solid samples, eg to prepare the sample material for analysis purposes. For example, sewage sludge is homogenized prior to analysis with rotor-stator homogenizers. But other biological materials such as tissue of plant or animal origin are homogenized by means of rotor-stator homogenizers for their analysis. Rotor-stator homogenizers are used, for example, in the sample preparation of brain tissue for the detection of prions and thus play an important role in the detection of transmissible spongiform encephalitis. However, due to the high rotational speeds, it is usually the case that the samples are even spilled out of the reaction vessel. This leads to losses as well as to a reduction in yield and carries the risk of contamination, which is undesirable not only with infectious material but also, for example, with toxic and / or radiolabelled material. Further problems are lack of reproducibility, too low homogenization efficiency, poor automation, cumbersome and less efficient transfer of reagents, solutions, homogenate, etc.

The present invention was therefore based on the object to provide a device that allows a more efficient homogenization of liquid or solid samples. The solution to this problem is to provide the objects and methods mentioned in the claims.

The present invention reduces or prevents the spattering of sample material, thereby reducing the risk of contamination. In addition, the invention also offers the advantage of achieving higher yields and better reproducibility. This is especially important for diagnostic purposes. Furthermore, according to the invention, due to the nature of the rotor-stator homogenizer, the reaction vessel or homogenization vessel, their connection by means of a connection system and / or the corresponding combination of rotor-stator homogenizer, reaction vessel and / or connection system and corresponding processes a faster and more efficient Homogenization achieved, for example, the surprising surprising the efficient rotor-stator homogenization of a microorganism, such as Escherichia coli (E. coli) allows. The invention also offers the possibility of inexpensively combining reusable rotor-stator homogenizers with disposable vessels, if appropriate including homogenization particles and / or digestion reagents, ie "homogenization kits." This option opens up the lucrative option of marketing "homogenization kits" to customers with corresponding rotor-stator homogenizers. The possibility of this cost-effective combination and the increased efficiency and efficiency of homogenization is advantageous over the known disposable rotor-stator homogenizers and the complete disposable systems. The present invention relates to a device for the homogenization of inhomogeneous samples, wherein a rotor-stator-homogenizer is connected to a reaction vessel in a manner that the ejection of the sample from the vessel is avoided or at least reduced and / or the rotor-stator Homogenizer defined with the reaction vessel is connected, so that, for example, the homogenization efficiency and yields are increased, reduces the risk of contamination and / or the automation and disposal of waste, such as the complete unit of rotor-stator homogenizer, reaction vessel, and Connection system after homogenization and removal Homogenate, be relieved. In addition, surprisingly, a higher reproducibility of homogenizations can be achieved by the inventive, defined and standardized connection of the rotor-stator homogenizer with the reaction vessel. The defined anchoring through the connection between the rotor-stator homogenizer and the reaction vessel thus obviously has the advantage that the homogenization process becomes more reproducible. In addition to the connection system according to the invention of a rotor-stator homogenizer with a reaction vessel, a further aspect of the invention in an apparatus or a method, wherein homogenization surfaces on the rotor-stator homogenizer, on the reaction vessel, on the connection system or in the form of Homogenisationspartikeln are present.

An additional aspect of the invention is an apparatus which is a rotor-stator homogenizer which is used not only for homogenization but also for the transfer of material such as liquids, reagents, homogenate, etc.

The connection of a rotor-stator homogenizer with a reaction vessel is achieved by a connection system.

An exemplary structure of a rotor-stator homogenization system according to the invention is shown in FIG. A coupling (1) connects the rotor-stator-homogenizer (2) with a drive. The coupling, which is open at the top, can be closed by means of a closure, for example a lid, possibly also a membranous closure, a septum, a fleece, etc., in order to avoid contamination and to enable adequate disposal. Substances can be added or removed via the open coupling or the rotor-stator homogenizer which is open at the top. For example, homogenate can be removed from the reaction vessel via a pipetting process. The rotor-stator-homogenizer (2) consists of a stator (3) and a rotor (4). Preferably, openings can be integrated. Besides a possible upper opening for the circulation of air at a position outside the reaction vessel (5a), it is preferable to integrate a lower lateral opening for the circulation of solution (6). In a particular embodiment, the upper opening for the circulation of air outside the reaction vessel (5a) is dispensed with. Instead, a corresponding opening in the rotor-stator homogenizer is introduced at a position within the reaction vessel (5b). Homogenate, which emerges from this opening (5b), so can no longer splash out of the reaction vessel and to Contaminations and losses. The openings can be located exclusively in the stator (3), exclusively in the rotor (4) or in both components. For a rotor that does not consist of a hollow, cylindrical component, but is based on a solid rod, openings are generally dispensed with. The rotor-stator homogenizer (2) has homogenization regions (7). By means of at least one connection system (8), the rotor-stator homogenizer (2) is connected to the reaction vessel (9).

The rotor-stator homogenizer, the reaction vessel and the connection system are made of suitable materials. For example, made of plastics or metal. Particularly preferred is the combination of a rotor-stator homogenizer made of metal with a reaction vessel and a connection system made of plastic or glass. The rotor-stator homogenizer can thus be used several times if necessary. The homogenization vessel and possibly the connection system can be used for centrifuging the sample or for further process steps such as pipetting, incubations, etc., and finally disposed of in a sterile, low-contamination or contamination-free manner. If the rotor-stator-homogenizer is not used several times, in particular in the case of rotor-stator-homogenizers, which are made of plastic, this can be processed together with the reaction vessel and the connection system and can also be disposed of in a sterile and low-contamination or contamination-free manner.

In the following, exemplary embodiments of the connection system are listed. Preferred connection systems are constructed and consist of components and elements which cause a seal so that no material exits the reaction vessel or the rotor-stator-homogenizer during the homogenization or during subsequent process steps or the exit is reduced and / or a stable and / or defined positioning of the rotor-stator homogenizer in the reaction vessel is brought about. An additional aspect of connection systems according to the invention may be a device used for the transfer of material, for example liquids, reagents, homogenate, etc. FIG. 2A shows an embodiment in which the connection system (8) is placed on the peripheral edge of the reaction vessel (9) and the rotor-stator homogenizer (2) is preferably enclosed as tightly as possible by a cylindrical region of the connection system (14) is and thus a guide or lock for the rotor-stator-homogenous isator and the outlet of solution or homogenate from the reaction vessel reduced or is prevented. In FIG. 2B, a guide or catch (15) is provided in the connection system, which is in direct contact with the inner surface of the reaction vessel, so that the connection system is solid or defined in the reaction vessel. For example, the connection system may be firmly connected to the reaction vessel like a lid. The opening of the connection system may also have a closure or lid. The guide (15) can also be located outside the upper edge of the reaction vessel (Figure 2C). The connection system can also have a separation unit (11) (separation units are explained in greater detail below, see also FIG. 5). In addition, the connection system may be provided with openings (16) for pressure equalization or pipetting. These openings can be equipped with membranes, valves or lids or closures. It is also conceivable that the inner guide is constructed so that it also serves as a guide for the rotor-stator-homogenizer (Figure 2D). Furthermore, a pipette tip, a guide for a pipette or pipette tip, or an adapter for a pipette (17) can be integrated into the connection component. In a further embodiment, the connection system is provided with a border (18) which offers additional protection against the splash of solution or homogenate and further reduces contamination risks (FIG. 2E). In addition to a simple insertion of the rotor-stator homogenizer in the leadership of the connection system, it is also possible to use more rigid connections (Figure 2F, 19), such as snaps, bayonet or threaded connections. Such compounds can be used not only in the connection between the connection system and rotor-stator-homogenizer, but also in the connection between the connection system and the reaction vessel. With such compounds, for example, the vertical position of the rotor-stator homogenizer is also determined relative to the reaction vessel. The vertical position of the rotor-stator homogenizer can also be achieved by a stopper (Figure 2G, 20). This stopper can be part of the rotor-stator homogenizer, ie, for example, be firmly connected to this. It is likewise conceivable for the stopper to be fastened to the rotor-stator homogenizer by means of a more flexible, yet rigid connection, for example a snap lock, bayonet closure or a threaded connection, for example by a locking screw (FIG. 2G, 21). In a further embodiment (Figure 2H), the stopper is conical and designed the connection system appropriately.

In order to increase the tightness between the rotor-stator-homogenizer and the connection system or the reaction vessel and the connection system, seals and sealing systems can also be used. As shown in Figure 21, for example, a sealing ring (22), e.g. an O-ring, which is preferably in a guide (23) or depression, are used for this purpose. In conjunction with the stopper of the rotor-stator homogenizer so a correspondingly high density is achieved. In a further embodiment (Figure 2J), the connection system is constructed so that the vertical axis of symmetry of the rotor-stator homogenizer (dotted line) is not congruent with the vertical axis of symmetry of the reaction vessel (dashed line), so that the angle α is not equal to 90 ° is. For example, the introduction of an angle, e.g. between 0.5 ° and 85 °, between 1 ° and 40 °, between 2 ° and 20 °, or between 3 ° and 10 °. In a further embodiment, the angle can be changed manually, but preferably by an external device (drive unit), by means of a flexible shaft, for example through a hose. A flexible shaft can also be made by the flexible connection of a stable cylindrical shaft with a stable lid. As a flexible compound, for example, correspondingly flexible plastics come into question.

Runs the rotor-stator-homogenizer obliquely relative to the vertical axis of a reaction vessel, this has surprisingly the advantage that the formation of a vortex or "Homogenisationstrichters" in the center of the reaction vessel in the solution or in the homogenate is prevented or reduced, in the rotor-stator-homogenizer runs "empty". This prevents the homogenization efficiency from being reduced. Furthermore, thus excessive foaming is suppressed, which adversely affects the yield. Foaming may result in denaturation of the sample, negatively affecting later analyzes. In addition, foam formed by the transfer, for example by a pipetting process or decantation, the sample from the reaction vessel into other vessels difficult. The losses in the sample during the transfer of a "foamed" solution are considerable, and according to the invention these losses can be reduced. In a further embodiment (Figure 2K), the connection system is constructed so that the vertical axis of symmetry of the rotor-stator homogenizer (dotted line) at a defined position, outside the central vertical axis of the reaction vessel (dashed line) is located. This also prevents a vortex from forming in the center of the reaction vessel in which the rotor-stator homogenizer is "empty", with the consequences described above.

In a further embodiment, the azentrisch mounted rotor-stator-homogenizer additionally in the horizontal plane, preferably circular, are moved. This process can be done manually, but preferably via a corresponding drive, so that a corresponding reproducibility i is ensured. The connection system has, for example, an insert (FIGS. 2L, 24). This insert allows, for example, circular movements of the rotor-stator homogenizer, which surprisingly improves the homogenization.

In a further embodiment, differently sized inserts or elements of the connection system, for example, as shown in Figure 2L, 24, used to flexibly connect different sized rotor-stator-homogenizers and reaction vessels. As described above (FIG. 2F / G), the predetermined connection between the rotor-stator homogenizer and the reaction vessel can also determine the relative height. It is also possible to change this relative height during the homogenization process. For example, repeated up and down movements are conceivable, which are done manually, but preferably via a corresponding drive, so that a corresponding reproducibility is maintained. These movements in the vertical plane can be combined with movements in the horizontal plane as well as angle changes manually, but preferably by a corresponding drive. Surprisingly, these movements in a plane or in the room mean that material that otherwise escapes the homogenization process is still homogenized, and this in particular in the case of an automated and therefore reproducible process.

In general, the embodiments may be provided with a lid or closure (25), which is shown by way of example in FIG. 2L. The various aspects of the embodiments can be combined with each other according to the invention.

In a further embodiment, the surfaces of the rotor-stator homogenizer and / or the reaction vessel are such that the homogenization effect or homogenization efficiency increases surprisingly significantly. This is achieved through the use of homogenization surfaces. Homogenization surfaces are characterized in that surfaces are used with a rough texture, i. for example, that the surface of a surface, in particular a smooth surface, is increased. For example, rough surfaces may be generated by sanding, grinding, drilling, sand blasting, ultrasound, etching or other methods. It is also conceivable, for example, that in the case of plastic articles, the corresponding injection molding tools are not polished, but if necessary, in particular provided with rough surfaces. The plastic articles thus produced may then have rough surfaces. It is also possible to apply particles to surfaces, so that, for example, a surface is formed as with a sandpaper. It is also possible to use free particles (homogenization particles). This means that homogenization surfaces can also be homogenization particles. Homogenization particles are solid particles, for example glass, silica, titanium, magnetic particles, etc., which are added before or during the homogenization process and lead to an unexpected increase in the homogenization effect.

In FIG. 3, homogenization surfaces are located on the inside of the reaction vessel (10a), in the rotor-stator homogenizer (10b), in particular in the region which is introduced into the reaction vessel, preferably on the outer surface of the stator, and / or in the form of homogenization particles (10c ), available. For example, it is possible with a system that does not homogenization or no digestion of microorganisms without Homogenisationsoberflächen homogenize or unlock with Homogenisationsoberflächen, for example, homogenization particles, microorganisms. Surprisingly, the combination of homogenization surfaces with homogenization reagents gives unexpectedly high homogenization rates. Homogenization reagents are reagents or solutions that contain urea, detergents, organic solvents, chaotropic salts, enzymes, etc. As detergents, for example, sodium dodecyl sulfate, tween Detergents (eg Tween 20), Triton detergents (eg Triton-X 100), etc. are used. As organic solvents, for example, dimethylsulfoxide, methanol, ethanol, etc. are used. Chaotropic salts are, for example, guanidinium salts (eg guanidinium hydrochloride), NaCl or other salts (in high concentrations), etc. Detergents and chaotropic salts may also be combined; for example, as guanidinium dodecyl sulfate. Enzymes are, for example, hydrolases, in particular proteases (proteinase K), muramidases, etc.

Homogenization particles can also be used for parallel or subsequent separation processes. For example, silica particles can be used to purify nucleic acids.

The use of magnetic particles offers the advantage of carrying out subsequent magnetically assisted separation processes or extraction processes directly in the reaction vessel without having to add magnetic particles in a further process step. It is possible to reversibly magnetize the rotor-stator homogenizer, so that the rotor-stator homogenizer can be used for the reversible binding of the magnetic particles. This can be of great advantage in extraction or purification steps, in particular in their automation. Besides particles, in the rotor-stator homogenizer, the reaction vessel or the connection system, membranes, filters, nonwovens, chromatography units, etc. may be integrated, which also allow the performance of separation processes. Furthermore, openings may be provided in the rotor-stator homogenizer, the reaction vessel or the connection system which allow pipetting operations or separation processes. These openings may contain septa, membranes, filters or other penetrable or permeable systems.

The rotor-stator-homogenizer shown in Figure 4 can be used as a pipette for the addition of solutions, reagents, etc. and for the removal of homogenate and for carrying out separation steps. For this purpose, the coupling (1), preferably formed so that it can be connected to a corresponding pipetting system (26), for example, a connection to a pumping system of a robot or a manual pipette.

In order to use the rotor-stator homogenizer as a pipette, preferably no openings or closable in the rotor-stator homogenizer are used. For pressure equalization also openings in the connection system (5c) or the Reaction vessel (5d) may be used with membranes, septa, valves or lids or other closures.

If only the rotor (3) is used as a pipette, i. Liquid or homogenate moves inside the rotor, preferably openings in the stator (4) are integrated. If both the rotor (3) and the stator (4) used as a pipette, i. Liquid is moved inside a hollow rotor and / or between rotor and stator, openings are preferably integrated into the rotor.

In a particular embodiment, the tip of the rotor-stator homogenizer is tapered, e.g. conical, shaped (7b). Here, the tooth-shaped structure of the Homogenisationsbereiche remains. The pipette tip may be on the rotor or on the stator. Preferably, the tip is not tooth-shaped but completely closed to optimally absorb the liquid. Surprisingly, the use of the rotor-stator homogenizer as a pipette leads to an increase in the yield and reduction of the risk of contamination. Furthermore, the number of process steps is reduced. This facilitates automation, standardization, simplifies and speeds up processes and reduces costs. In a further embodiment (FIG. 5), separation units for carrying out separation processes, extraction processes or

Chromatography method provided (11). There is at least one separation unit. But it can also be more integrated. The separation units can be located in the rotor-stator homogenizer (11a), in the reaction vessel (11b) and also in the connection system. The separation units can consist of a simple membrane or a fleece that is anchored in a corresponding holder (12). The separation units can also be constructed more complex. For example, two membranes (12) in respective holders can enclose a material (13) which can be used for extraction or chromatographic processes. For example, these may be hydroxyapatite, silica, sepharose, phenylsepharose, etc.

In FIG. 6, the proof that the use of a device according to the invention prevents the occurrence of contaminations, in contrast to the prior art, is illustrated in an exemplary embodiment (exemplary embodiment 1). Using a device according to the invention results in no contamination after a treatment time of one minute (picture left). It becomes that in FIG. 2B used illustrated connection system. Here, the connection system is firmly connected as a lid with the reaction vessel. By contrast, the device according to the prior art (without a connection system according to the invention) already shows a clear contamination after a treatment time of 15 seconds (picture on the right). For the experimental verification of the prevention of contamination by means of devices according to the invention, 700 μl of a 2% aqueous Coomassie Brilliant Blue G 250 (CBB) solution are transferred into 2 ml reaction vessels from Eppendorf (Hamburg, Germany). Using a Miccra 8 rotor-stator homogenizer from ART modern laboratory technology (Müllhausen, Germany) is homogenized at room temperature at a rotational speed of 33,000 min ^'1 . The proof of a possible splashing of the CBB solution and a concomitant contamination of the environment is demonstrated by means of absorbent Whatmann paper from Schleicher & Schuell. The paper is in each case mounted cylindrically around the reaction vessel and the rotor-stator-homogenizer.

FIG. 7 shows in an exemplary embodiment (exemplary embodiment 2) the dependence of the slopes of the release kinetics of the lactate deoxygenase from Escherichia coli DH 5α (E. coli) on the rotor-stator homogenization system used. The gradients are determined in linear areas. The gradients are given in seconds x liters / units [sec * L / U]. The release of the lactate dehydrogenases from E. coli reflects the homogenization or the digestion of this microorganism. Smooth reaction vessels and smooth rotor-stator homogenizers (gHgRS), rough reaction vessels and smooth rotor-stator homogenizers (rHgRS), smooth reaction vessels and rough rotor-stator homogenizers (gHrRS) as well as rough reaction vessels and rough rotor-stator are used in the experiments - Homogenizers (rHrRS) used. In rough rotor-stator homogenizers, the outer surface of the stator is roughened by sanding. The reaction vessels are roughened inside by drilling with a drill of appropriate diameter. The experiments clearly show the higher homogenization efficiency when using rough surfaces in contrast to smooth surfaces. Embodiment 3

For homogenization and any subsequent purification of genomic DNA, homogenization particles, in particular magnetic particles, are used as homogenization surfaces. The Dynabeads® DNA DIRECT ™ Universal kit (product No. 630.06 from Dynal® Biotech) is used to perform the experiment. Tissue of a vertebrate, especially 50 to 100 mg of muscle meat is placed in a reaction vessel. 1, 5 ml, 2 ml, 15 ml and 50 ml reaction tubes are used. Preferably 2 ml reaction vessels from Eppendorf are used. 200 μl of digestion solution (lysis buffer of the Dynabeads® DNA DIRECT ™ Universal kit) are placed in the reaction vessel. On the one hand, homogenizations are carried out in which Dynabeads are located in the lysis buffer, on the other hand, homogenisations are carried out in which there are no Dynabeads in the lysis buffer. Using a Miccra rotor-stator homogenizer is homogenized at speeds of rotation of 10,500 to 39,000 min ^'1 , preferably at 30,000 min ^{' 1} , at room temperature or cooled, for example by ice. The homogenization time is 2 to 60 seconds, preferably 5 seconds. On the one hand, this is done with connection systems according to the invention, in particular according to Figure 2 B, C and D, worked on the other hand without connection systems. When using connection systems according to FIG. 2C and FIG. 2D, an adhesive strip is used as the cover or closure for the openings (16, 17) during the homogenization. The homogenates of the experimental preparations are either further processed for the subsequent purification of genomic DNA (see below) or treated as follows. The homogenates are removed by pipetting and their volume determined. If necessary, it may be necessary to cut off the pipette tip at the lower end with a suitable pair of scissors or a knife so that an opening is formed which is sufficiently large to prevent clogging by pieces of tissue. This measure may also be necessary in other process steps and other embodiments. This is followed by a ten minute centrifugation with 10,000 g at room temperature. The volume of the supernatant is determined. The absorbance of the supernatant at 260 nm is determined after adequate dilution with water. Furthermore _| For example, after adequate dilution with water, Bradford protein concentration is determined (Bradford, MM (1976) Anal. Biochem., 72, 248-254). The absorption at 260 nm and the protein concentration based on the amount of tissue used are used as a measure of the efficiency of the homogenization of a tissue (Vitzthum, F. (2000) Development and investigation of automation-appropriate physical-mechanical disintegration method for a nucleic acid-based, human medical infection diagnostics, Fraunhofer IRB Verlag , ISBN 3-8167-5582- 8th). In addition, the protein yield, the product of the protein concentration and the volume of the supernatant, is determined.

On average, the inventive approaches with Dynabeads or compound systems and in particular their combination based on the amount of tissue used, higher absorptions, higher protein concentrations, larger homogenate volumes, and / or total protein yields as comparable approaches without Dynabeads and / or compound systems.

This is due, inter alia, to the fact that in the connection systems according to the invention the splashing of the sample out of the reaction vessel is reduced or prevented. It shows that the connection systems not only reduce or prevent the risk of contamination, but also increase the volume yield and the reproducibility of the results. This manifests itself above all with a long duration of the homogenization, for example 60 seconds when 2 ml reaction vessels are used. In addition, since more material remains in the reaction vessel and thus remains longer in the homogenization process and is better homogenized, the efficiency of the homogenization increases.

The efficiency of the homogenization is also surprisingly enhanced by the homogenization particles, in particular in combination with the compound systems. Especially with a short duration of homogenization, for example from 2 to 20 seconds when 2 ml reaction vessels are used and when using connection systems, the efficiency of homogenization in the presence of homogenization particles is greater than without. For the purification of genomic DNA in the homogenates without Dynabeads the homogenate is transferred as usual in another reaction vessel in which are the Dynabeads, which were previously removed from the lysis buffer. This step costs time, which is economized according to the invention and advantageous in the approaches with Dynabeads in the digestion solution and during homogenization. As a reaction vessel, the reaction vessels are used, which were also used in the homogenization, preferably 2 ml reaction vessels from Eppendorf, to the following process steps under comparable

Conditions to perform. The transfer of the homogenate is carried out in such a way that the dynabeads distribute themselves sufficiently in the homogenate. In the approaches with connection systems according to Figure 2 C or Figure 2 D, the rotor-stator-homogenizer and the respective connection system during the procedure is not removed. The transfer of material takes place here via the connection systems. This saves time.

All mixtures are incubated at room temperature for 5 to 10 minutes. After this incubation, the reaction vessels are positioned in or on a Dynal Magnetic Particle Concentrator (MPC). Preferably, the magnet is initially at the bottom of the reaction vessels. This is particularly important in the approaches in which the rotor-stator homogenizer is still in the reaction vessel to achieve the most complete collection of Dynabeads. When the Dynabeads have gathered, the magnet can be moved to the side, pulling the dynabeads slightly to the side. This facilitates the removal of the supernatant by pipetting. Preferably, the rotor-stator homogenizer is displaced slightly upwardly from its original position near the bottom of the reaction vessel, which it occupies during homogenization, but without completely removing it. After at least 1 to 2 minutes, the supernatant is removed by careful pipetting. At the bottom of the reaction vessel is a brown, gel-like complex of DNA and Dynabeads. The following steps will be taken to ensure that this complex is not destroyed. After the supernatant is removed, the reaction vessel is removed from the Dynal MPC and 200 μl wash solution (10 mM Tris-HCl, pH 7.5, 150 mM LiCl, 0.1 mM EDTA) added by pipetting. The reaction vessel is then placed back on or in the Dynal MPC. After the solution has become clear, the supernatant is removed. This washing procedure is repeated once. Thereafter, the complex of DNA and DynabeacJs in 20 to 50 ul resuspension solution (10 mM Tris-HCl, pH 8.0) is resuspended. For the resuspension is preferably incubated at 65 ⁰ C for 5 minutes. The reaction vessel is placed in the Dynal MPC and after about 30 seconds, the supernatant is removed. After a corresponding dilution with water, the determination of the absorption at 260 nm determines the DNA concentration (Vitzthum, F. and Bernhagen, J. (2002) SYBR Green I: An ultrasensitive fluorescent dye for double-stranded DNA quantification in solution and Other applications; Recent Res. Devel., Anal., Biochem., 65-93, ISBN 381-7895-054-5).

On average, the approaches according to the invention with Dynabeads or compound systems and in particular their combination provide higher DNA concentrations than comparable approaches without Dynabeads and / or connection systems. Particularly preferred here is the use of one of Connection systems of Figure 2 C and Figure 2 D. Very particularly preferred is a connection system according to Figure 2 C without filter (11) but with opening (16). Embodiment 4

This embodiment describes by way of example the transfer of material through corresponding connection systems. A Miccra rotor-stator-Horηogenisator is

I used. The reaction vessels used are 50 ml Greiner centrifuge tubes. The upper portion of the centrifuge tubes is separated approximately at the 20 ml markings. Lid of centrifuge tubes are provided with a hole in the center of the lid or a bore outside the central axis for the Miccra 8 rotor-stator homogenizer and at least one other bore outside the central axis for a pipette tip. Using hot glue (article number 539500 from LUX tools) using a hot glue gun (KCK2002 from King Craft, Müller & Partner GbR), the rotor-stator homogenizer is connected to the respective covers with corresponding holes, so that systems as shown in FIG. Figure 2 K or combinations are constructed. Preferably, the rotor-stator homogenizer is introduced so that it is directed obliquely in the center of the bottom tapered reaction vessel with closed screw and the end of the rotor-stator-homogenizer with the crushing areas approximately in the middle just above the tapered bottom the reaction vessel comes to rest. The lid is attached to the reaction vessel with hot glue. The attachment of the rotor-stator homogenizer outside the central axis and / or with a corresponding inclination (see Figure 2J) shows a reduced risk that the rotor-stator homogenizer will run "empty", thus increasing the efficiency of homogenization. At least one small piece of tissue, for example a total of 50 to 200 mg of muscle meat, is added to the reaction vessel through the pipetting orifice, and the digestion solution is added through the pipetting opening by pipette until the digestion solution has a volume in the region of the labeling of the reaction vessel of 5 to 15 ml Digestion solution, for example, phosphate-buffered, physiological saline pH 7.4 (PBS) is used.

During the homogenization of 15 to 120 seconds at room temperature without cooling closes at a rotational speed of 30,000 min ^"1 either an adhesive strip as a kind of cover, the pipette, or it is according to figure 2 D a pipette tip (17) therein. By this Pipetting or pipette tip is still after homogenization Detergent Triton X 100 added to a final concentration of 1% ι. To distribute the detergent homogeneously, we further homogenize the tissue homogenate for a short moment at minimum rotational speed. Alternatively, the rotor-stator homogenizer is removed from the drive and the reaction vessel is gently shaken. The addition of detergent; leads to a more complete, more efficient homogenization of the tissue than without detergent and for the release of in particular membrane-bound substances; for example, membrane-bound proteins. In contrast to an addition of the detergent in the first homogenization, the addition of the detergent only after the first homogenization, the foaming is less.

In such a process, embodiments of the connection system according to FIG. 2 C and D are to be used preferably, since these, in contrast to connection systems without openings (16) or pipette tips (17J), a simple, fast, contamination-free or low-contamination, lossless or low-loss and automation-compliant addition and also allow removal of material without separating the reaction vessel from the connection system or rotor-stator homogenizer from the connection system. Optionally, this pipette tip has a filter (11) as shown in Figure 2C. The tissue homogenate is removed via a pipette tip. Additional PBS is added via the pipette tip, homogenized briefly again and the homogenate is pipetted off. This process is repeated twice more. This increases the protein yield (see below). The total volume of the homogenate from these steps is determined. This is followed by a ten-minute centrifugation with 10,000 g at room temperature. The volume of the supernatant is determined. The absorbance of the supernatant at 260 nm is determined after adequate dilution with water. Furthermore, after a reasonable dilution with water, the Bradford protein concentration is determined. The absorbance at 260 nm and the protein concentration based on the amount of tissue used are used as a measure of the efficiency of homogenization of a tissue. In addition, the protein yield, the product of the protein concentration and the volume of the supernatant, is determined.

On average, the approaches according to the invention with compound systems which are suitable for the transfer of material provide greater homogenate volumes and, based on the amount of tissue used, higher total absorptions, the product of homogenate volume and absorption per inserted amount of tissue and / or higher Total protein yields, the product of homogenate volume and protein concentration per amount of tissue used, as comparable approaches without linkage systems. Embodiment 5 Here, a rotor-stator homogenizer according to Figs. 8a and 8b is used for transferring material. A correspondingly cut pipette tip (7b) is glued into a hollow rotor according to FIG. 8a. In this case, for example, superglue gel from Tesa is used as an adhesive. Openings of the rotor-stator homogenizer are made, for example, by means of adhesive strips and / or adhesive except for the openings for the circulation of air in the rotor (5a) and stator (5a *) and the opening in the stator 5b / 6 for the circulation of air and / or liquid sealed. A connection system according to FIG. 2 B is used. In a further embodiment, as shown in FIG. 8a, this connection system has an opening (5d) whose function will be discussed below.

In a reaction vessel, water and salad oil are added. At minimum rotation speed, homogenize for a short moment. The resulting suspension is removed via the rotor-stator homogenizer. This happens for example via the rotor. A pipette or pipette tip or other equivalent system for generating pressure changes (26), for example, a vacuum for taking out material or an overpressure for adding material that can be accurately connected to the rotor or the rotor-stator homogenizer, is inserted into the rotor (arrow 27), optionally between the rotor and the stator, so that the opening in the rotor (5a) is closed. For clarification, the process performed in Figure 8b is shown in section of the stator and the top view of the rotor and the top view of the system for generating pressure changes (26). In the exemplary embodiment, a suitable pipette tip is cut to size and modified with hot glue in such a way that a shape corresponding to FIGS. 8a and 8b is produced. The opening (5a) in the rotor can alternatively also be closed from the outside, for example by a plug. For this purpose, however, the openings in the rotor (5a) and in the stator (5a *) must be in the same position to shoot the opening in the rotor (5a) through the opening of the stator (5a *). If a negative pressure is generated by means of a pipette or a corresponding system, the suspension passes into the rotor via the pipette tip on the rotor (7d), in which case the via the opening on the stator (5a *) acting air pressure or via any openings in the connection system (16) presses them into the rotor. The rotor-stator homogenizer is then used like a pipette tip, ie removed from the reaction vessel and the suspension contained in the rotor transferred to another reaction vessel. Alternatively, the suspension is removed from the reaction vessel via the rotor-stator-homogenizer remaining in the reaction vessel.

With correspondingly large volumes and solutions, suspensions and homogenates with appropriate viscosity, it is possible with the device according to the invention and the method according to the invention to use the rotor or the rotor-stator homogenizer without a pipette tip (7b) for the transfer of solutions. However, the use of a pipette tip is preferred, since some solutions can only be pipetted with it at all and / or less or no residual volume remains in the reaction vessel and thus yields are increased with a pipette tip on the rotor-stator homogenizer. Preferably, we fed via the rotor material to the reaction vessel or removed. Especially when the rotor-stator-homogenizer is used as a kind of pipette tip. If the rotor-stator-homogenizer is separated again after the transfer of material from the system that produces pressure changes, this is an advantage as it reduces the risk of material leakage. However, a further possibility according to the invention for the transfer of material also consists in the use of corresponding systems for generating pressure changes (26) which fit precisely around the outside of the stator. These are plugged or pushed over the stator, so that the opening on the stator (5a *) is fired from the outside. Optionally, in this case, the gap between the rotor and stator is sealed. Due to the pressure conditions, it may be useful here to use stators that have no openings 5b / 6 (Figure 8a) use. In a further embodiment, stators are used with the openings 5b / 6 (FIG. 8a) using connection systems which likewise have at least one opening 5d (FIG. 8a). During homogenization, the rotor-stator homogenizer is in a position where the opening or openings of the stator coincide with the opening or openings of the connection system so that the circulation of air or material is possible. By changing the position of the stator relative to the connection system, at least one opening of the stator can be closed. This is exploited when removing material. The rotor-stator homogenizers according to the invention and processes which are suitable for the transfer of material, in particular in combination with the compound systems according to the invention, allow a simpler and faster transfer of material, ie in this case a suspension of water and salad oil and / or im Means larger yields of this homogenate compared to comparable homogenizations in which the homogenate in a conventional manner, for example by decantation or pipetting is removed, especially when no inventive compound systems are used. Embodiment 6

Rotor-stator homogenization of plant material in conjunction with a reaction vessel with separation unit for filtration of the homogenate. Here, material from the NucleoSpin® Plant XL kit from Macherey & Nagel is used. Leaves of a plant, for example leaves of a penny-seed tree, are transferred into a NucleoSpin® filter unit, ie a reaction vessel approximately corresponding to FIG. 5. The drain of the NucleoSpin® Filter Units is closed. A rotor-stator homogenizer is connected via a connection system according to Figure 2 C without a Separätionseinheit (11) with the NucleoSpin® Filter Unit. The "Buffer C1" of the kit is pipetted in via the opening 16 of the connection system The volume of "Buffer C1 added" corresponds to the volume recommended in the test description of the kit for an appropriate amount of plant material. The opening 16 is closed with an adhesive strip. At room temperature is homogenized at a rotational speed of 30,000 min ^"1 from 5 seconds to 2 minutes. The tape is removed after the homogenization and per mg of sample 80 ul of RNase A solution of the kit are added by pipette through the opening 16. It is at 60 ° C is incubated for 30 minutes The NucleoSpin® Filter Units drain is removed by overpressure applied via the rotor-stator homogenizer or the connection system with simultaneous closure of appropriate openings in the connection system and / or rotor-stator homogenizer, the homogenate is pressed through the NucleoSpin® Filter Unit The filtered homogenate is collected in a reaction vessel and further processed, if desired, according to the protocol of the kit Using the compound system according to the invention and the combination according to the invention with a separation unit, in particular a filtration unit it in the reaction vessel, the otherwise necessary change of Reaction vessels avoided. On average, this shortens the process duration and increases the yield.

The methods and apparatuses listed in the description and the exemplary embodiments can be combined according to the invention and can advantageously be used correspondingly to the respective requirements in a corresponding combination.

Claims

Claims:

1. A device for rotor-stator homogenization inhomogeneous samples, characterized in that a. a rotor-stator homogenizer and a reaction vessel are connected to each other via a connection system and / or b. Homogenization surfaces and / or c. a rotor-stator homogenizer suitable for the transfer of material can be used.

2. Apparatus according to claim 1, characterized in that the connection system connects the rotor-stator-homogenizer with the reaction vessel so that the rotor-stator-homogenizer is in a defined position in the reaction vessel.

3. Device according to one of claims 1 or 2, characterized i characterized in that the connection system connects the rotor-stator homogerizer with the reaction vessel so that the escape of material is prevented or reduced.

4. Device according to claims 1 to 3, characterized in that the

I

Rotor-stator homogenizer, the reaction vessel or the connection system made of plastic or metal.

5. Device according to claims 1 to 3, characterized in that the reaction vessel or the connection system of glass steal.

6. Device according to one of claims 1 to 5, characterized in that the connection system connects the rotor-stator-homogenizer with the reaction vessel so that the rotor-stator-homogenizer is perpendicular but outside the central, vertical axis of the reaction vessel.

7. Device according to one of claims 1 to 6, characterized in that the connection system connects the rotor-stator-homogenizer with the reaction vessel so that the vertical axis of symmetry of the rotor-stator-homogenizer is not parallel to the vertical axis of symmetry of the reaction vessel.

8. Device according to one of claims 1 to 7, characterized in that the rotor-stator-homogenizer and the reaction vessel are movable relative to each other.

9. Device according to one of claims 1 to 8, characterized in that the rotor-stator-homogenizer, the connection system and / or the reaction vessel have openings for the transfer, the addition or the removal of solutions, reagents and homogenate.

10. Device according to one of claims 1 to 9, characterized in that the reaction vessel, the connection system and / or the rotor-stator-homogenizer contains at least one separation unit for carrying out separation, extraction or chromatography.

Method for Homogenizing Inhomogeneous Samples By ₍ characterized in that a device according to any one of claims 1-10 is used.

12. The method according to claim 11, further characterized in that digestion reagents are used.

13. The method according to claim 11 and 12, further characterized in that magnetic homogenization particles are used.