Abstract
A gel electrophoresis apparatus for single cell gel electrophoresis, including a chamber for receiving a gel electrophoresis buffer, a functional cover for closing the chamber, at least one pair of electrodes for generating a homogeneous electric field in the chamber and at least one retaining element for receiving and positioning at least one support plate. The at least one retaining element positions the at least one support plate in the homogeneous electric field generated by the at least one pair of electrodes.
Claims
1. A gel electrophoresis device for single cell gel electrophoreses, said gel electrophoresis device comprises: a chamber for receiving a gel electrophoresis buffer, a lid for closing the chamber, at least one electrode pair for producing a homogeneous electrical field in the chamber, and at least one retaining element for receiving and positioning at least one carrier plate, wherein the at least one retaining element is configured to position the at least one carrier plate in the homogeneous electrical field which is produced by the at least one electrode pair, wherein the gel electrophoresis device comprises at least one measuring electrode for measuring an electrical field, said measuring electrode being different from the electrodes of the at least one electrode pair, and wherein the gel electrophoresis buffer can flow freely around the carrier plate.
2. The gel electrophoresis device according to claim 1, wherein the carrier plate comprises a planar surface.
3. The gel electrophoresis device according to claim 1, wherein at least one retaining element is arranged in the functional lid and/or on the base of the chamber.
4. The gel electrophoresis device according to claim 1, wherein the at least one retaining element is designed as the measuring electrode for measuring an electrical field.
5. The gel electrophoresis device according to claim 1, wherein the at least one retaining element comprises a head part and at least one latch-in section for the fixation of at least one carrier plate.
6. The gel electrophoresis device according to claim 5, wherein the at least one retaining element comprises a first latch-in section for the fixation of a first carrier plate and a second latch-in section for the fixation of a second carrier plate, a spacer between the first latch-in section and the second latch-in section and an anchor.
7. The gel electrophoresis device according to claim 1, wherein at least one barrier is arranged on a side of a wall of the chamber for producing a laminar buffer flow.
8. The gel electrophoresis device according to claim 7, wherein the at least one barrier comprises at least one continuous, horizontally running slot-like passage and/or several shorter slot-like, vertically running openings and/or tubular passages.
9. The gel electrophoresis device according to claim 1, wherein the gel electrophoresis device comprises a carrier element with at least one retaining element and the carrier element is positionable in the chamber.
10. The gel electrophoresis device according to claim 9, wherein the carrier element comprises an incorrect-rotation safeguard and wherein a corresponding incorrect-rotation safeguard is arranged on the base of the chamber.
11. A gel electrophoresis system for single cell electrophoreses with a high reproducibility, wherein the gel electrophoresis system comprises: a gel electrophoresis device according to claim 1 and in integrated means for the temperature control and/or an integrated means for cooling and/or heat generation and/or an integrated means for the buffer circulation and/or an integrated voltage generating device and/or an integrated mains connection device and/or integrative software and/or a subsequently arranged, integrated and automated analysis device for the quantification of the results and/or a digital interface for the further processing of the results.
12. A carrier plate for the positioning in a gel electrophoresis device according to claim 1, as well as for receiving at least one gel, wherein the carrier plate comprises a planar surface and a polyester film with a hydrophilic surface is deposited on said planar surface and/or the planar surface is treated with a hydrophilic layer.
13. The carrier plate according to claim 12, wherein the carrier plate comprises an edge region with at least one opening for receiving and positioning on at least one retaining element.
14. The carrier plate according to claim 13, wherein the carrier plate comprises an edge region with two openings and wherein at least one of the openings with regard to its shaping differs from the other openings.
15. The carrier plate according to claim 12, wherein the carrier plate comprises an alkali-resistant material with breakage strength of at least 15 to 20 Newton.
16. The carrier plate according to claim 12, wherein the carrier plate comprises a spacer element.
17. A handling frame for a carrier plate according to claim 12, wherein the handling frame comprises at least one fastening means for receiving the carrier plate, wherein the fastening means comprises a base part and a pin-like prominence.
18. A method for reproducibly carrying out a comet assay can whilst using the gel electrophoresis system according to claim 11, which comprises the following steps: depositing the gel spot with the cells to be examined onto a carrier plate with the help of a single-channel or multi-channel pipette; positioning the carrier plates on the at least one retaining element of the chamber; closing a functional lid; incubation of the carrier plates with the deposited gel spots in an alkaline environment for DNA unwinding; selecting the electrophoresis program and initiating the control of the electrophoresis parameters by way of integrative software, for producing a homogeneous filed over the at least one carrier plate; opening the functional lid and removing the carrier plate; bringing the carrier plate into a staining solution; drying gel spot in ethanol; removing the carrier plate from the staining tank and bringing the carrier plate into a washing solution; optional drying of the gel spot on the carrier plate; manual or semi-automated or automated microscopic evaluation of the gel spot.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The subject matter of the invention is hereinafter explained in more detail by way of preferred embodiment examples, which are represented in the accompanying drawings. In each case are represented schematically in:
(2) FIG. 1a is a plan view of a carrier plate with round openings at the corners;
(3) FIG. 1b is a plan view of a carrier plate with oval openings at the corners;
(4) FIG. 2a shows a retaining element for carrier plates with a laterally flattened spacer;
(5) FIG. 2b shows a retaining element for carrier plates as in FIG. 2a, a non-flattened side of the spacer being represented;
(6) FIG. 2c shows a retaining element for carrier plates with a polygonal spacer;
(7) FIG. 2d shows a retaining element for carrier plates with spacers flattened at two sides;
(8) FIG. 3 shows two carrier plates, which are arranged above one another in a sandwiched manner, fixed by way of four retaining elements;
(9) FIG. 4a shows a gel electrophoresis device with 32 carrier plates, which are fixed in the functional lid;
(10) FIG. 4b shows a gel electrophoresis device with 32 carrier plates, which are located in the chamber;
(11) FIG. 5a and FIG. 5c show a detail of the chamber wall with fastening means for the electrodes, which are arranged in pairs;
(12) FIG. 5b shows a fastening means for electrodes, which are arranged in pairs;
(13) FIG. 6a shows a handling frame with pin-like prominences on triangular base parts, said prominences being at the corners;
(14) FIG. 6b shows a handling frame with pin-like prominences on round base parts, said prominences being at the corners;
(15) FIG. 7a shows a pipetting aid with a pipetting block and a frame and openings;
(16) FIG. 7b shows a plan view of the pipetting aid with a pipette tip;
(17) FIG. 7c shows a view of the longitudinal side of the pipetting aid with a metal frame and carrier plate;
(18) FIG. 7d shows a view of the transverse side of the pipetting aid with a pipetting block and carrier plate;
(19) FIG. 8a shows a barrier with tubular passages;
(20) FIG. 8b shows a barrier with slot-like passages;
(21) FIG. 9a, FIG. 9b, FIG. 9c are plan views of a carrier plate;
(22) FIG. 10a and FIG. 10b show a carrier element with carrier plates;
(23) FIG. 10c is a plan view of an electrophoresis tank;
(24) FIG. 10d is an electrophoresis tank with an inserted carrier element;
(25) FIG. 11 show a cross section through the electrophoresis tank in the closed condition;
(26) FIG. 12a and FIG. 12b show a carrier plate with openings and spacer elements;
(27) FIG. 12c shows an electrophoresis tank with carrier plates, which are positioned on the functional lid; and
(28) FIG. 13 an electrophoresis tank with rail-like retaining elements.
DETAILED DESCRIPTION OF THE INVENTION
(29) In principle, the same or analogous parts are provided with the same references numerals in the figures.
(30) FIGS. 1a and 1b show a plan view of a rectangular carrier plate 1a with a film surface 2 and with an edge region 4 which is provided with markings 5 on a longitudinal and a transverse side, the markings defining a grid for the gel spots that are to be deposited onto the film. The film surface is a planar surface of the carrier plate, on which a polyester film with a hydrophilic surface is deposited. The carrier plate with the planar surface can alternatively be treated with a hydrophilic layer.
(31) The openings 3 of the carrier plate 1a, which are at the corners, are represented in a round manner in FIG. 1a, and in FIG. 1b as oval openings. Of course, further embodiments of the openings 3, 3 such as, for example, square or rectangular openings or openings of an arbitrary shape are possible. The carrier plate 1 is bevelled in the edge region of one of the openings 3a, which is at the corner. This bevelling positively matches a retaining element, which is specially matched to this and characterises the orientation of the carrier plate 1 in the chamber 7a, which is to say determines the orientation of the carrier plate 1 in the chamber 7a.
(32) FIG. 2a shows a retaining element for the fixation of the carrier plates 1 in an electrophoresis chamber, thus in a chamber for receiving the gel electrophoresis buffer. The retaining element 6 includes a head part 6a, a latch-in section 6b for the fixation of a first carrier plate 1, a latch-in section 6b for the fixation of a second carrier plate, a spacer 6c, which on using two carrier plates, keeps these at a distance, and an anchor 6d, which can be anchored in the functional lid of a gel electrophoresis device or however placed or positioned in the base of the chamber of a gel electrophoresis device.
(33) The spacer 6c is represented in FIG. 2a with a flattened side. FIG. 2b shows the oppositely lying arched side of the spacer 6c.
(34) FIG. 2c shows a spacer 6c in a polygonal embodiment with four flattened sides, and in FIG. 2d an embodiment of a spacer 6c with two flattened and two arched sides is represented.
(35) The head 6a and/or the spacer 6c, 6c, 6c, 6c of a retaining element 6 can be designed as a measuring electrode.
(36) On using more than one retaining element 6, for example, one of the spacers with regard to its embodiment can differ from other spacers. Such a spacer, for example, can be adapted to an opening and/or its surrounding of a carrier plate, which differs from the other openings and/or surrounding of the carrier plate. An orientation of the at least one carrier plate 1 in the functional lid or the base of the electrophoresis chamber and which is always the same can be ensured by way of this.
(37) FIG. 3 shows two carrier plates 1 and four retaining elements 6 in the assembled condition. The two carrier plates 1 are assembled above one another by way of four retaining elements 6. On account of its openings, the first carrier plate 1 is fixed in the first latch-in sections 6b of the four retaining elements and the second carrier plate 1 by way of its openings is fixed in the second latch-in sections of the four retaining elements 6. Spacers 6c ensure a constant distance between the carrier plates 1. In this embodiment example, the two carrier plates are fixed in a manner such that the films 2 with the gel spots deposited thereon are arranged face-to-back. In an alternative embodiment, the carrier plates 1 are fastened in the retaining elements 6 in a manner such that the films 2 with the gel spots lie facing one another (face-to-face).
(38) FIG. 4a shows an electrophoresis tank 7 including an electrophoresis chamber 7a, thus a chamber for receiving a gel electrophoresis buffer, a functional lid 7b and a grip 7c. The electrophoresis chamber 7a in the interior and on a longitudinal side in each case includes two electrode pairs 8 and 8, which run in parallel and which are of an alkali-resistant material such as platinum-iridium. The electrode pairs are designed as electrode wires and are fixed in the electrophoresis chamber 7a via fastening means 9. The fastening means 9 simultaneously serve as a connection between the electrode and electricity source, in particular as a conductive connection.
(39) When the functional lid 7b is closed, then the electrode pairs 8 and 8 are located at the height of the intermediate space between the carrier plates 1 or the films, which are with the gel spots and which are located on these plates.
(40) The functional lid 7b of the electrophoresis tank 7 on its side that is directed to the electrophoresis chamber 8a includes twelve retaining elements, which are fixed on the lid 7b. The retaining elements are arranged in three groups of four retaining elements 6. Two carrier plates are fixed face-to-face, i.e., with the film or the surface treated with the hydrophilic layer and the gels spots located thereon facing one another, per group of retaining elements. Openings for the retaining elements, into which openings the anchor of a retaining element can be introduced and fastened are incorporated or arranged in the lid of the electrophoresis chamber.
(41) The functional lid moreover includes a grip 7c for opening and closing the electrophoresis chamber 7a. The functional lid 7b is connected to the electrophoresis chamber 7a at two transverse sides by way of hinge joints 10, 10. These hinge joints 10, 10 can be arrestable.
(42) Moreover, the electrophoresis tank 7 on a longitudinal side of the electrophoresis chamber 7a and on a longitudinal side of the functional lid 7b includes parts of a closure mechanism 11, 11a, 11a. The closure mechanism can include a magnet, for example.
(43) FIG. 4b shows an electrophoresis tank 7 including an electrophoresis chamber 7a, a functional lid 7b and a grip 7c. The electrophoresis chamber 7a in the interior and on a longitudinal side includes two electrodes 8 and 8, which run in parallel and are of an electrochemically noble material such as, for example, platinum-iridium. The electrode wires are fixed in the electrophoresis chamber 7a via fastening means 9. The fastening means 9 simultaneously serve as a connection between the electrode and electricity source.
(44) In contrast to FIG. 4a, the carrier plates 1 shown in FIG. 4b are fastened in or on the base of the electrophoresis chamber by way of retaining elements. For this, openings, into which the anchor of the retaining elements 76 is insertable and can be fastened are incorporated in the base of the electrophoresis chamber. The retaining elements can also be in the chamber in an unfastened manner and be placed onto the base of the chamber.
(45) FIG. 5a shows a detail of a longitudinal side of the electrophoresis chamber of one of the two FIG. 4a or 4b with the two electrodes of the electrode pairs 8 and 8, which run in a parallel manner on the longitudinal side and with a fastening means 9 and 9 for the electrodes 8 and 8. FIG. 5c shows a similar detail of a longitudinal side of the electrophoresis chamber 7a. The electrode pairs 8, 8 are covered by a protective element 80 in order to prevent an unintended contact with an electrode pair 8.
(46) FIG. 5b shows an enlarged detail of the longitudinal side of the electrophoresis chamber of one of the two FIG. 4a or 4b, with a fastening means 9. The electrode wires 8 and 8 are led through a passage through a transverse wall, which lies transversely or perpendicularly to the longitudinal side and from there are led through a recess in the fastening means 9 and are fixed. The fastening means 9 leads from the inner side of the longitudinal side through an opening onto the outer side of the longitudinal side and there is connected to an electricity source.
(47) FIG. 6a shows a handling frame 12, which at the corners includes triangular prominences 12b, thus a prominence with a triangular cross section, wherein the prominences 12b at the middle each include a pin-like extension 12a that latchably or positively fit into openings of the carrier plate, which are provided for these. After the latching, the carrier plate is fixedly connected to the handling frame 12.
(48) FIG. 6b shows a further possible embodiment of a handling frame 12 with round prominences 12a at the corners, each with pin-like extension 12b, which is central in this.
(49) FIG. 7a shows a perspective view of a pipetting aid 13 with a pipetting block with 96 conical openings. The pipetting block in the region of a longitudinal side is not releasably, but rather is fixedly, connected to a frame 13a. In the region of transverse sides running transversely to the longitudinal side, the pipetting block is shorter than the frame that surrounds it, by 0.5 to 1 cm. In other words: the pipetting block with regard to one dimension is smaller than the frame.
(50) FIG. 7d shows a plan view of one of the transverse sides of the pipetting aid. The pipetting block 13b is not releasably connected to the frame. An intermediate space is located between the pipetting block 13b and the lower part of the frame.
(51) FIG. 7b represents the upper side of the pipetting aid. The frame 13a is not designed in a continuous manner as is shown in FIG. 7a, but in the region of the lower side of the pipetting aid 13 includes a recess in the size of a carrier plate 1. The pipetting aid with the recess can be placed onto the carrier plate and fixed there in a slip-proof manner, for pipetting the gel spots onto the film.
(52) FIG. 7c is a lateral view of the longitudinal side of the pipetting aid and shows the frame with a lateral recess, by which means the pipetting block is visible.
(53) FIG. 8a shows a barrier 14 with tubular passages 14a for the control of the buffer flow, the barrier being able to be attached, for example, in a side wall of an electrophoresis chamber. The buffer flow which is led out of a tube (not shown), for example, from a heating and/or cooling system is led through the tubular openings, broken up and thereby homogenised.
(54) FIG. 8b shows an alternative barrier 15 for the control of the buffer flow, with slot-like passages 15a, which are aligned perpendicularly to the base of the electrophoresis chamber when the barrier is attached to a side wall of the electrophoresis chamber.
(55) A plan view of the hydrophilic side of a carrier plate 1 is shown in FIG. 9a. FIG. 9b shows the side of the carrier plate 1 that is away from the hydrophilic side. A polyester film 2, which extends up to into the edge region 4 of the carrier plate 1, is arranged on the hydrophilic side of the carrier plate 1. The corner regions of the carrier plate 1 are free of polyester film 2. Four spacer elements 20, which each enclose an opening 3 in a hollow-cylinder-like manner, are arranged on the side of the carrier plate 1 that is opposite to the polyester film 2. Here, the two spacer elements 30 in the edge region 4 of the transverse side of the carrier plate 1 with markings 5 are spaced further from one another than the two spacer elements 30, which are arranged in the edge region 4 of the opposite transverse side of the carrier plate 1. The asymmetrical arrangement of the hollow-cylinder-like spacer elements 30 permits an orientation-specific positioning of the carrier plate 1 on the functional lid 7b and/or in the chamber 7a.
(56) The hollow-cylinder-like spacer elements 30 include a slot that extends essentially along a longitudinal axis of the spacer element 30. The spacer element 30 obtains elastic or resilient characteristics by way of this slot, and these elastic characteristics permit a repeated, destruction-free positioning of the carrier plate 1 on the retaining element 6.
(57) An alternative embodiment of a carrier plate 1, as is shown in FIG. 9a, is represented in FIG. 9c. Here, the carrier plate 1 includes continuous openings 3, which, similarly to FIGS. 9a and 9b, are surrounded by a hollow-cylinder-shaped spacer element 30. In contrast to this, the openings 3 of the carrier plate 1 in FIGS. 9a and 9b are not continuous, but are designed as blind holes. As is shown in FIG. 9c, the openings 3 and the spacer elements 30 can be arranged symmetrically in the corner region of the carrier plate 1.
(58) FIGS. 10a and 10b show a carrier element 16 with three carrier plates 1, as are shown in FIGS. 9a and 9b. The carrier element 16 includes twelve retaining elements 6. which are designed in a manner such that they receive and position the three carrier plates 1, thus fix them relative to the carrier element 16. FIG. 10b shows the carrier plate 1 in the positioned condition on the carrier element 16. The spacer elements 30 embrace the retaining elements 6 and the carrier plates 1 can be positioned in the homogenous, electrical field of the electrode pairs 8 in the chamber 7a with the help of the retaining elements 6. The polyester film 2, thus the hydrophilic side of the carrier plate 1 is arranged on the side that is away from the carrier element 16. The carrier element 16 includes an incorrect-rotation safeguard 162 that permits a specific, orientation-selective arrangement of the carrier element 16 in the chamber 7a. The carrier element 16 includes two carrier grips 161 that simplify the arranging of the carrier elements 16 in the chamber 7a of the electrophoresis tank 7.
(59) FIG. 10c shows a gel electrophoresis device in the opened condition, thus with an open functional lid 7b. Three carrier plates 1, which are fixed or positioned on respective retaining elements 6, are positioned on the functional lid 7b. An incorrect-rotation safeguard 72, which corresponds to the incorrect-rotation safeguard 162 of the carrier element 16, is arranged on the base of the chamber 7a.
(60) FIG. 10d shows the carrier element 16 with carrier plates 1 that are positioned thereon, the carrier element being inserted into the electrophoresis tank 7, in particular into the chamber 7a, as is shown in FIG. 10c. The incorrect-rotation safeguard 162 of the carrier element 16 engages into the incorrect-rotation safeguard on the base of the chamber 72 in an essentially exactly fitting manner. The specific, orientation-selective arrangement of the carrier element 16 in the chamber 7a is ensured by way of this. Six carrier plates 1 are therefore positioned on the electrophoresis tank 7 with the help of corresponding retaining elements 6.
(61) FIG. 11 shows the cross section through an electrophoresis tank in the closed condition, similarly as in FIG. 10d, but with a closed functional lid 7b. Three carrier plates 1 are fixed or positioned on the functional lid 7b with the help of retaining elements 6. Three further carrier plates 1 are fixed or positioned on the carrier element 16 with the help of retaining elements 6. The carrier element 16 is arranged on the base of the chamber 7a. The carrier element 16 is arranged in the chamber 7a in an orientation-specific manner with the help of the incorrect-rotation safeguard 162 on the base of the chamber 7a. The carrier plates 1 are positioned in the electrophoresis tank 7 in a face-to-face arrangement, wherein the electric field, which is produced by the electrode pairs 8, 8, is most homogeneous in the region of the hydrophilic surface of the carrier plate 1. As is shown in FIG. 11, the carrier plates 1 are arranged essentially at the height of the electrode pairs 8, 8 when the carrier element 16 with the positioned carrier plates 1 is inserted into the chamber 7a, and the function lid 7b with the positioned carrier plates 1 is closed.
(62) The base of the chamber 7a has a slight descent, as is to be seen in FIG. 11. The gel electrophoresis buffer can be drained out of the chamber 7a in a simpler manner by way of this, which is to say that it can be exchanged essentially without any residue.
(63) FIGS. 12a and 12b show a view of a carrier plate 1 with openings 3 and spacer elements 30. Similarly as in FIG. 9, the openings 3 and the spacer element 30 are arranged in the edge region 4 of the carrier plate 1. The openings 3 have a keyhole-shaped cross section with a first region and a second region. The first region has a larger diameter than the second region, wherein the first region is adjacent to the second region. In other words: the first region is essentially circular and the second region is designed as an indentation that connects to the first region. Four spacer elements 30 are arranged in the edge region 4 of the opposite transverse sides, on the side of the carrier pate 1 which is away from the hydrophilic side (see FIG. 12b). The spacer elements 30 serve for the simple stacking of carrier plates 1 above one another, wherein the spacer elements 30 of a first carrier plate 1 can be placed in a space-saving manner in the edge region 4 of a second carrier plate 1 without damaging samples/gel spots which are deposited on the hydrophilic side.
(64) FIG. 12c shows an electrophoresis tank 7 with carrier plates 1, which are positioned on the functional lid 7b, as is shown in FIGS. 12a and 12b. The retaining element 6, which is fixed or fastened on the functional lid 7b, is designed in an essentially pin-like or mushroom-like manner and can engage into correspondingly congruent, which is to say correspondingly designed openings 3 of the carrier plate 1, wherein the latch-in section 6b of the retaining element 6 is positioned in the openings 3 of the carrier plate 1 in the positioned condition of the carrier plate 1.
(65) The head part 6a is designed in a manner such that it fits through the first region of a keyhole-shaped opening 3 of the carrier plate 1. The head part can be pushed through this first region of the openings 3. The latch-in section 6b, which has a smaller diameter than the head part 6a, can subsequently be positioned or latched in, in the second region of the keyhole-shaped opening 3 of the carrier plate 1.
(66) In FIG. 13, the retaining element 6 is designed in a rail-like manner with a groove as a latch-in section 6b, wherein the edge region 4 of the carrier plate 1 can be inserted or positioned in the groove of the retaining element. The head part 6a of the retaining element 6 is designed as a terminating surface.
