CN112710720B - Single cell gel electrophoresis glue spreading method and device - Google Patents
Single cell gel electrophoresis glue spreading method and device Download PDFInfo
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- CN112710720B CN112710720B CN202011492729.7A CN202011492729A CN112710720B CN 112710720 B CN112710720 B CN 112710720B CN 202011492729 A CN202011492729 A CN 202011492729A CN 112710720 B CN112710720 B CN 112710720B
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Abstract
The invention discloses a single cell gel electrophoresis glue spreading method and a single cell gel electrophoresis glue spreading device. The invention provides a single cell gel electrophoresis gel spreading device, which comprises a gel spreading device, a movable plate, a film and a shell; one surface of the glue spreader is arranged in a groove, a plurality of cylindrical bulges are arranged in the groove, and bottom glue is spread in the bottom glue space between the cylindrical bulges; the groove surface of the glue spreader is closely overlapped with the movable plate and is an upper flat surface and a lower flat surface; the film is wrapped by the glue spreader and the movable plate in a U shape, and then is superposed into an upper flat plane and a lower flat plane, and the U-shaped bottom of the film is provided with an opening; the shell is a hollow cuboid with an upper bottom surface opening and a lower bottom surface opening, the shell is sleeved outside the thin film and is tightly connected with the thin film, the lower bottom surface opening of the shell is communicated with the U-shaped bottom opening of the thin film, and one end of the movable plate is dissociated outside the bottom surface opening of the shell. The method and the device can help to lay out the gel with fixed thickness and can prevent the occurrence of degumming condition in subsequent experimental operation.
Description
Technical Field
The invention relates to a gel spreading method and a device, in particular to a single cell gel electrophoresis gel spreading method and a device.
Background
Single Cell Gel Electrophoresis (SCGE), also known as comet assay, was first proposed by Ostling et al in 1984 and used to detect radiation-induced DNA double strand breaks under neutral conditions. In 1988, singh et al established an alkaline single-cell gel electrophoresis technique which can not only detect single-strand and double-strand breaks in DNA, but also detect alkaline instability sites, DNA cross-linking and incomplete excision repair sites, and the like. SCGE is widely applied to numerous fields such as genetic toxicology, radiation biology, oncology, pathology, environmental ecology, epidemiology, environmental and professional population biological monitoring and the like.
The conventional glue spreading method adopts a sandwich glue spreading method: uniformly spreading 100 mu l of normal melting point agarose gel on a glass slide, flattening the gel surface, and curing in a refrigerator at 4 ℃ for 10 minutes; uniformly mixing 25 mul of cell suspension and 75 mul of low-melting-point agarose gel, uniformly paving the mixture on the layer 1 gel, and placing the gel in a refrigerator at 4 ℃ for 10 minutes to solidify; mu.l of low-melting agarose gel was spread evenly on top of the layer 2 gel and cured in a freezer at 4 ℃ for 10 minutes. At present, a double-layer glue spreading method without using top glue or a single-layer glue spreading method only containing cell low-melting-point glue is mostly adopted in a single-cell gel electrophoresis test, and a finished glue spreading glass slide is provided.
The sample after spreading the glue is also required to be subjected to the steps of cracking, rinsing, unwinding, neutralizing, electrophoresis, dyeing and observation. In all processes, glue spreading is the foundation and the most important experimental operation, and no matter the sandwich glue spreading method, the double-layer glue spreading method or the single-layer glue spreading method, the problems of uneven glue spreading and degumming in the operation process after glue spreading cannot be solved, and the development of the single-cell gel electrophoresis technology is influenced due to the slow glue spreading process.
Disclosure of Invention
In view of the above problems, the present invention provides a method and an apparatus for spreading gel by single cell gel electrophoresis, which can help to spread gel with a fixed thickness and prevent the occurrence of degumming in the subsequent experimental operations.
The invention provides a single cell gel electrophoresis gel spreading device, which comprises a gel spreading device, a movable plate, a film and a shell;
one surface of the glue spreader is arranged in a groove, a plurality of columnar bulges are arranged in the groove, and bottom glue is spread in the bottom glue space between the columnar bulges; the groove surface of the glue spreader is tightly superposed with the movable plate to form an upper flat surface and a lower flat surface;
the film wraps the glue spreader and the movable plate in a U shape, and then the glue spreader and the movable plate are superposed to form an upper flat plane and a lower flat plane, and an opening is formed in the U-shaped bottom of the film and used for drawing out the movable plate;
the shell is a hollow cuboid with an upper bottom surface opening and a lower bottom surface opening, the shell is sleeved outside the film and is tightly connected with the film, the lower bottom surface opening of the shell is communicated with the U-shaped bottom opening of the film, and one end of the movable plate is dissociated outside the bottom surface opening of the shell.
In the invention, the gel sample to be paved dripped above the movable plate is filled in the glue spreader and a gap divided between the shell and the film along with the downward extraction of the movable plate when the device is vertically arranged along the glue spreading direction.
In the device, one end of the glue spreader at the top of the U-shaped opening of the film is provided with a glue pouring hole for spreading bottom glue in the bottom glue space between the cylindrical bulges of the glue spreader;
the raw materials of the bottom layer glue are normal melt glue.
The bottom layer glue is a normal melt adhesive known in the field, experimental materials known in the field can be purchased from commercial sources, the normal melt adhesive melts at about 90 ℃, and can form glue at more than 30 ℃; the gel sample is melted by low-melting point glue at about 65 ℃ and can be formed into gel at the temperature below 30 ℃; the primary purpose of laying the bottom layer glue is to enable a gel sample (including low-melting-point glue of a cell sample) to be flat and tightly adhered, and prevent the gel sample from falling off from a glue laying device; the invention is provided with a bulge which can not only increase the area of the bottom layer glue and a glue spreader, but also prevent the relative sliding of the glue, and aims to prevent the gel sample from falling off.
In the device, the glue spreader comprises a plurality of blocks, two ends of the groove surface of each glue spreader are respectively provided with a notch at the top end and the bottom end along the glue spreading direction of the device, and the U-shaped bottom of the film corresponding to the notch at the bottom end of each glue spreader is provided with an opening communicated with the notch;
the movable plate is comb-shaped, each comb tooth of the movable plate is closely superposed with the glue spreader through a bottom notch of the glue spreader, and the movable plate is free from the U-shaped bottom opening of the film and the bottom opening of the shell except for the part of the comb tooth of the movable plate.
In the invention, the glue spreader can be composed of 1-10 blocks, preferably 5 blocks.
In the device, the top end of the groove surface of the glue spreader at the U-shaped opening of the film is in a wedge shape; a wedge-shaped gap is formed between the wedge-shaped top end of the glue spreader and the movable plate, and the detachable occupation block is seamlessly filled in the gap.
In the invention, the occupation block can be a solid block, a hollow block or a wedge-shaped sheet matched with the wedge-shaped gap; when the glue spreader comprises a plurality of blocks, the glue spreader is matched with the glue spreader according to the arrangement of the notch formed at the top end of the glue spreader, so that the wedge-shaped gap is seamlessly filled.
In the above device, the free edges of the U-shaped opening end of the film are respectively provided with a solid block, and the solid blocks are clamped on the opening of the upper bottom surface of the housing.
In the above apparatus, the solid block is made of plastic.
In the above device, the shape of the cylindrical protrusion is cylindrical and/or square-cylindrical.
In the above apparatus, the thickness of the movable plate is 0.05mm to 0.2mm.
In the above device, the movable plate is made of rigid plastic or metal;
the glue spreader and the shell are made of plastics;
the film is made of soft plastics.
The invention also provides a method for spreading glue by using the device.
The invention has the following beneficial effects:
1. the cylindrical bulges are arranged on the glue spreader, so that the contact area between the bottom glue and the glue spreader is increased, and the degumming condition in the experimental process is prevented.
2. The low-melting point glue containing the sample is sucked into the glue spreading device along with the movement of the movable plate and is covered on the bottom glue, the thickness of the sample is the thickness of the slice, and the thin and uniform sample layer can be obtained by controlling the thickness of the movable plate.
3. The bottom layer glue can be prepared in advance or made into a reagent form, the glue spreading process can be completed only by pulling out the thin sheet, the operation is simple, and the automation is suitable.
4. The wedge-shaped gap is formed between the wedge-shaped gap and the movable plate, the gap is filled by the occupied block in a seamless mode before glue spreading operation, the occupied block is removed during glue spreading operation, a certain amount of sample glue is added into the wedge-shaped gap, the thick end of the movable plate is pulled out of the movable plate, and the sample glue can be automatically filled into the gap left after the movable plate is pulled out.
5. The solid block (such as a small plastic block) fixed at the opening end of the film can be clamped on the shell, and after the glue spreading is finished, the plastic blocks at the two ends of the film are simultaneously pulled, so that the glue spreader is easy to take out.
Drawings
FIG. 1 is a schematic side cross-sectional view of the apparatus of the present invention.
Fig. 2 is a schematic side sectional view of the apparatus of the present invention in operation, with the arrows pointing in the direction of the movable plate and the membrane, respectively.
FIG. 3 is a schematic side cross-sectional view of the glue spreader, movable plate and placeholder of the apparatus of the present invention.
FIG. 4 is a schematic side cross-sectional view of the membrane and housing, respectively, of the device of the present invention.
FIG. 5 is a schematic three-dimensional view and a schematic front view of a glue spreader of the apparatus of the present invention; fig. 5 (a) is a three-dimensional schematic view, and fig. 5 (b) is a schematic view as viewed from the front.
Fig. 6 is a three-dimensional schematic view (left view) and a front view schematic view (right view) of a movable plate of the device.
FIG. 7 is a three-dimensional schematic view of the device film, housing, and placeholder, respectively, of the present invention.
The labels in the figure are as follows:
1 glue spreader, 2 movable plates, 3 films, 4 shells, 5 bottom glue spaces, 6 wedge-shaped spaces, 7 occupied blocks, 8 bottom glue, 9 sample glue and 10 glue pouring holes.
FIG. 8 is a graph of the results after gel spreading electrophoresis of commercially available finished consumables (cells not in the same focal plane).
FIG. 9 is a graph showing the results of gel application electrophoresis of the apparatus of the present invention.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The invention will be further described with reference to the accompanying drawings, but the invention is not limited to the following examples.
Example 1 Single cell gel electrophoresis gel spreading method and device
1. Single cell gel electrophoresis spreads mucilage binding and puts
As shown in figure 1, the single cell gel electrophoresis gel spreading device of the invention comprises a gel spreader 1, a movable plate 2 with the thickness of 0.05 mm-0.2 mm, a thin film 3 and a shell 4 (the materials are plastics, the materials do not cause cell nucleus damage and do not have autofluorescence, and the materials of the embodiment adopt medical PP materials) which are tightly overlapped from inside to outside in sequence;
the bottom of the glue spreader 1 is provided with a plurality of column-shaped (specifically, column-shaped, square column-shaped or other shapes) protrusions for increasing the contact area between the bottom glue 8 (normal melting point glue, such as normal melting point agarose) and the glue spreader 1 and preventing the occurrence of the 'degumming' condition, and the bottom glue space 5 in the glue spreader 1 is used for spreading the bottom glue 8. The bottom layer glue 8 can be added into the bottom layer glue space 5,4 ℃ through the glue pouring hole 10 on the glue spreader 1 before the experiment and is solidified and prepared; or be prepared into commercial consumables in advance and stored at 4 ℃.
The movable plate 2 can be a layer of hard plastic thin plate, the thickness of the movable plate 2 is 0.1mm, the glue spreader 1 matched with the movable plate is divided into 5 blocks (1-10 blocks can be designed as required), the other end of the divided movable plate 2 is thicker (1.0 mm) and is connected into blocks, the movable plate 2 is in a comb shape, the thick end of the movable plate 2 can be clamped on a notch reserved in the glue spreader 1, and the movable plate 2 can be conveniently dragged to be drawn out from the glue spreader 1. The glue spreader 1 and the movable plate 2 are combined into an upper flat plane and a lower flat plane.
One end of the glue spreader 1 is wedge-shaped, a wedge-shaped gap 6 is formed between the wedge-shaped end of the glue spreader and the movable plate 2, the gap is seamlessly filled by an occupying block 7 before glue spreading operation, the occupying block 7 is removed during glue spreading operation, a certain amount of sample glue 9 is added into the wedge-shaped gap 6, the thick end of the movable plate 2 is pulled to extract the movable plate, the sample glue 9 is automatically filled into the gap left after the movable plate 2 is extracted, and the thickness of the gap is 0.1mm.
The film 3 is a soft plastic film with the thickness of 0.05mm, the glue spreader and the movable plate are wrapped in a U-shaped mode, and the wedge-shaped gap 6 is located on one side of the U-shaped opening. The shell 4 is a hollow cuboid with an upper bottom surface and a lower bottom surface penetrating through the opening, the inner side of the shell 4 is closely connected with the film 3, and the inner side surface of the shell 4 is smooth to ensure the smoothness of the adhesive paving. The opening end of the film 3 is fixed on a small plastic block which can be clamped on the shell, after the glue spreading is finished, the plastic blocks at the two ends of the film are simultaneously pulled, and the glue spreader 1 is taken out.
The preparation method comprises the following steps: but movable plate 2 penetrates film 3, and the card is in the breach department that shop's gluey ware 1 was reserved, and film 3 parcel shop's gluey ware 1 and movable plate 2 are put into shell 4 from shell 4 jagged opening part, and the plastic block card of film 3 is fixed in the breach department that the shell was reserved, and space between shop's gluey ware 1 and the movable plate is put into to spacer 7. Through one glue filling hole in the glue spreader 1, the common molten glue (bottom glue) which is melted in boiling water bath and cooled to 50 ℃ is filled into the glue spreader, and is placed at 4 ℃ for heat preservation for 30 minutes.
2. The device is utilized to spread glue
Spreading glue according to the following steps:
the device of the present invention was placed vertically in an environment of 37 ℃ and 25. Mu.L of cell suspension (about 1.0X 10) 6 mL) was mixed with 75 μ L of low melting point agarose gel to form a sample gel 9, the occupied block 7 was removed, and 50 μ L of the sample gel was dropped into the wedge-shaped gap 6 formed between the glue spreader 1 and the movable plate 2. After all the sample glue is added, the movable plate 2 is drawn out downwards, the sample glue 9 moves downwards along with the drawing of the movable plate 2, gaps left when the movable plate 2 is drawn out are filled, and the thickness of the glue is just 0.1mm of that of the movable plate. And after the glue spreading is finished, placing the glue spreading device at 4 ℃ for 10 minutes to solidify the sample glue 9, simultaneously downwards drawing the plastic blocks at the two ends of the thin film 3, supporting the glue spreader 1 out of the shell 1 along with the downward movement of the thin film 3, and simultaneously stripping the thin film from the glue spreader to finish the glue spreading operation.
3. Applications of
Experiment for detecting peripheral blood lymphocyte DNA damage by alkaline SCGE:
1. preparation of reagents:
1% normal melting point agarose: 0.05g of normal melting point agarose +5.0mL of 0.2mol/L PBS, pH 7.2;
1% low melting point agarose: 0.05g of low-melting-point agarose +5.0mL of 0.2mol/L PBS, pH 7.2;
(agarose solution is heated to more than 95 ℃ for melting, 1 percent of agarose with normal melting point is filled at about 50 ℃, and 1 percent of agarose with low melting point can keep fluidity at more than 37 ℃)
Alkaline lysis solution: naCl 2.5mol/L, na 2 EDTA 100mol/L,1% sodium lauroyl sarcosinate, tris base20mmol/L,10% NaOH adjusted pH to 10.0; adding 1% Tration X-100, 10% DMSO just before use);
alkaline electrophoresis solution (NaOH 30mmol/L, na) 2 EDTA2.5mmol/L，pH≧13.0)；
The raw materials for the above reagents are commercially available.
2. Experimental procedures
According to the preparation method of the inner bottom layer glue of the 'single cell gel electrophoresis glue spreading device' in the embodiment 1, 3mL of 1% normal melting point agarose is filled into a glue spreader to be used as the bottom layer glue, the bottom layer glue is placed at 4 ℃ for 30 minutes to be solidified, and the front layer glue is used at 37 ℃ for 10 minutes.
Culturing and harvesting peripheral blood lymphocytes, adjusting cell density to 1.0 × 10 6 /mL，1μM H 2 O 2 The oxidant treatment was carried out for 5 minutes. At 37 ℃, adding 180 μ L of 1% low melting point agarose into 20 μ L of lymphocyte suspension after oxidation treatment, mixing uniformly to obtain sample gel, and dripping 50 μ L of the sample gel into a wedge-shaped gap formed by a bottom plate gel spreader and a movable plate according to the method for spreading the gel by the single cell gel electrophoresis gel spreading device in the embodiment 1. And after all the sample glue is added, the movable plate is downwards drawn out, the sample glue moves downwards along with the drawing of the movable plate, gaps left when the movable plate is drawn out are filled, and the thickness of the glue is just 0.1mm of that of the movable sheet. After the glue spreading was completed, the glue spreading device was left at 4 ℃ for 10 minutes to solidify the sample glue. And meanwhile, the plastic blocks at the two ends of the film are downwards drawn, the glue spreader is supported out of the shell along with the downward movement of the film, and the film is also peeled from the glue spreader to remove the top plate and the plastic film, so that the glue spreading operation is completed.
Soaking the sheet spreading device with the spread glue in the alkaline cell lysate precooled at 4 ℃ for 2 hours; taking out, soaking in 4 deg.C alkaline electrophoresis solution for unwinding for 30 min, with the electrophoresis solution exceeding the glue surface by 0.5cm; setting the power voltage of the electrophoresis apparatus to be 1V/cm, the current to be 300mA, and carrying out electrophoresis for 20 minutes. Soaking and cleaning with deionized water for three times, each time for 5 minutes; DNA dye solution Ethidium Bromide (EB) is used for dyeing for 20 minutes, and deionized water is used for washing for three times; and (5) observing and photographing under a fluorescence microscope.
3. Results
The device disclosed by the invention is used for spreading glue, is simple and convenient to operate, and can effectively prevent the occurrence of 'degumming'; the thickness of the sample glue is uniformly paved at the height of 0.1mm, and the cells are on the same horizontal plane under a microscope (figure 9), so that the influence of different focal planes of the cells (figure 8) on result analysis caused by too thick sample glue is avoided.
Claims (8)
1. The utility model provides a glue spreading device for single cell gel electrophoresis, which is characterized in that: the device comprises a glue spreader, a movable plate, a film and a shell;
one surface of the glue spreader is arranged in a groove, a plurality of columnar bulges are arranged in the groove, and bottom glue is spread in the bottom glue space between the columnar bulges; the groove surface of the glue spreader is closely superposed with the movable plate to form an upper flat surface and a lower flat surface;
the film wraps the glue spreader and the movable plate in a U shape, and then the glue spreader and the movable plate are superposed to form an upper flat plane and a lower flat plane, and an opening is formed in the U-shaped bottom of the film and used for drawing out the movable plate;
the shell is a hollow cuboid with an upper bottom surface opening and a lower bottom surface opening, the shell is sleeved outside the thin film and is tightly connected with the thin film, the lower bottom surface opening of the shell is communicated with the U-shaped bottom opening of the thin film, and one end of the movable plate is dissociated outside the bottom surface opening of the shell;
the glue spreader comprises a plurality of blocks, two ends of the groove surface of each glue spreader are respectively provided with a notch at the top end and the bottom end along the glue spreading direction of the device, and the U-shaped bottom of the film corresponding to the notch at the bottom end of each glue spreader is provided with an opening communicated with the notch;
the movable plate is comb-shaped, each comb tooth of the movable plate is tightly superposed with the glue spreader through a bottom end notch of the glue spreader, and the movable plate is free from the U-shaped bottom opening of the film and the bottom opening of the shell except for the comb teeth;
the top end of the groove surface of the glue spreader at the U-shaped opening of the film is in a wedge shape; a wedge-shaped gap is formed between the wedge-shaped top end of the glue spreader and the movable plate, and the detachable occupation block is seamlessly filled in the gap.
2. The apparatus of claim 1, wherein: the glue spreader is provided with a glue pouring hole at one end of the top of the U-shaped opening of the film, and is used for spreading bottom glue in a bottom glue space between the cylindrical bulges of the glue spreader;
the raw materials of the bottom layer glue are normal melt glue.
3. The apparatus of claim 1 or 2, wherein: and the free edges of the U-shaped opening end of the film are respectively provided with a solid block, and the solid blocks are clamped on the opening on the upper bottom surface of the shell.
4. The apparatus of claim 3, wherein: the solid block is made of plastic.
5. The apparatus of claim 1 or 2, wherein: the shape of the cylindrical bulge is cylindrical and/or square column.
6. The apparatus of claim 1 or 2, wherein: the thickness of the movable plate is 0.05 mm-0.2 mm.
7. The apparatus of claim 1 or 2, wherein: the movable plate is made of hard plastic or metal;
the glue spreader and the shell are made of plastics;
the film is made of soft plastics.
8. Method for applying glue with a device according to any one of claims 1-7.
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| CN202011492729.7A CN112710720B (en) | 2020-12-17 | 2020-12-17 | Single cell gel electrophoresis glue spreading method and device |
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Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5938909A (en) * | 1996-03-15 | 1999-08-17 | Guo; Rong | Cup-shaped vertical slab gel electrophoresis apparatus |
| ITMO20050319A1 (en) * | 2005-11-29 | 2007-05-30 | Scm Group Spa | DISTRIBUTOR DEVICE OF GLUE |
| CN101865877A (en) * | 2010-06-25 | 2010-10-20 | 西北大学 | Gel paving method by using single cell gel electrophoresis |
| CN103468570B (en) * | 2013-09-22 | 2014-09-03 | 江苏省农业科学院 | Glass slide device capable of facilitating single cell gel electrophoresis test |
| CN105021685B (en) * | 2014-04-17 | 2019-03-05 | 上海伯楷安生物科技有限公司 | A kind of shell of the gel electrophoresis pre-prepared colloid of anti-leakage |
| CN204903458U (en) * | 2015-06-29 | 2015-12-23 | 中国辐射防护研究院 | A slide glass box for unicellular gel electrophoresis |
| CN204855929U (en) * | 2015-06-29 | 2015-12-09 | 中国辐射防护研究院 | A anticreep slide glass for unicellular gel electrophoresis |
| CN209513720U (en) * | 2019-02-15 | 2019-10-18 | 济宁学院 | A kind of Ago-Gel gel maker |
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