CA2427488A1 - Gel extraction apparatus - Google Patents
Gel extraction apparatus Download PDFInfo
- Publication number
- CA2427488A1 CA2427488A1 CA 2427488 CA2427488A CA2427488A1 CA 2427488 A1 CA2427488 A1 CA 2427488A1 CA 2427488 CA2427488 CA 2427488 CA 2427488 A CA2427488 A CA 2427488A CA 2427488 A1 CA2427488 A1 CA 2427488A1
- Authority
- CA
- Canada
- Prior art keywords
- gel
- holding chamber
- chamber
- electrode
- sample collecting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 238000000605 extraction Methods 0.000 title description 8
- 238000001962 electrophoresis Methods 0.000 claims abstract description 11
- 229920000642 polymer Polymers 0.000 claims abstract description 8
- 238000011084 recovery Methods 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 5
- 239000012530 fluid Substances 0.000 claims description 4
- 239000004020 conductor Substances 0.000 claims description 3
- 239000008151 electrolyte solution Substances 0.000 claims 1
- 239000011556 non-electrolytic solution Substances 0.000 claims 1
- 239000000243 solution Substances 0.000 claims 1
- 239000000872 buffer Substances 0.000 abstract description 5
- 239000007853 buffer solution Substances 0.000 abstract 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract 1
- 238000002474 experimental method Methods 0.000 abstract 1
- 239000000499 gel Substances 0.000 description 69
- 241000894007 species Species 0.000 description 9
- 238000000034 method Methods 0.000 description 7
- 239000000463 material Substances 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 238000001502 gel electrophoresis Methods 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000013508 migration Methods 0.000 description 3
- 230000005012 migration Effects 0.000 description 3
- 238000005204 segregation Methods 0.000 description 3
- 238000011109 contamination Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000011810 insulating material Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000009791 electrochemical migration reaction Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44739—Collecting the separated zones, e.g. blotting to a membrane or punching of gel spots
Abstract
The device allows molecular specimens to be isolated from the gel by the use of an electrophoresis system. The system consists of an electrode containing cap and a gel extractor which is made up of a sample collecting chamber and gel holding chamber with a cutting edge. The cutting edge is used to excise the desired band containing the desired molecular species from a polymer gel.
Upon addition of the electrode containing cap to the gel extractor, the molecular species is isolated into the sample collecting chamber via electrophoresis by application of a potential across the gel layer. This forces the molecular specimen out of the gel and into a buffer solution in the sample collecting chamber. The buffer is extracted from the container with the molecular specimen as a ready-to-use reagent for other experiments.
Upon addition of the electrode containing cap to the gel extractor, the molecular species is isolated into the sample collecting chamber via electrophoresis by application of a potential across the gel layer. This forces the molecular specimen out of the gel and into a buffer solution in the sample collecting chamber. The buffer is extracted from the container with the molecular specimen as a ready-to-use reagent for other experiments.
Description
Background of the Invention This invention relates to electrophoretic methods and apparatus for the puriftcation and recovery of a specified fraction of biomolecular specimen from an electrophoretic medium, including but not limited to nucleic acids.
In research laboratories, it is often necessary to separate and isolate a particular molecular specimen, such as DNA or protein, from a mixture. This is often done by electrophoresis, which uses an electrical potential to move electrically charged particles through a polymer gel and separate different specimens based on a particular characteristic such as size, charge, or polarity. After separating the molecular specimen in the gel, it is sometimes reeluired to recover a particular sample of interest from the gel Prior devices or methods have addressed the purification, concentration, segregation, or transfer of molecular species from gel media. Some prior devices were developed for specific molecular species (nucleic acids or proteins).
U.S. Patent S,44S,723 (issued August, 1995, Camacho) is a blotting apparatus for transferring electrophoretically separated material from electrophoresis gels in a transfer stack to an immobilizing membrane. To recover the sample, additional steps will be required.
U.S. Patent 5,102,518 (issued April 7, 1992, Doering et al.) is a device for electroeluting and concentrating small amounts of proteins from sample gels for subsequent concentration or processing in a sequencing device with minimized sample handling and minimized disturbance. It elutes desired molecules from gels in a sample holder directly onto a membrane of a centrifuge concentrator or a sequencing membrane.
U.S. Patent 5,217,593 (issued June 8, 1993, I~acConnell) describes a process for purifying DNA and the like. It selectively applies an electrical potential along an inlet path for selectively moving a plasmid into an elution chamber, then along an outlet path into a collection chamber with an elution window at the outlet end.
U.S. Patent 5,384,022 (issued January 24, 1995, Rajasekaran) incorporates a method and apparatus for electrophoretic DNA band isolation and recovery with improved yields of recovered DNA. It includes a second well downstream in the migration path of the molecular specimen. A
cup, having a rim, is inserted into tlhe second well. Together with a membrane also placed in the second well, the molecular specimen is prevented from migrating past the cup.
The molecular specimen migrates into the cup and. is aspirated.
U.S. Patent 6,146,511 (issued November 14, 2000, Slater et al.) is a 2-step electrophoretic method focussing on the purification of a nucleic acid sample by reducing the concentration of contaminants relative to the concentration of desired nucleic acid in the nucleic acid sample.
Several electrophoresis-based prior instruments were designed with specific devices for cutting/ excising and removing/recovering separated specimens from gel media.
U.S. Patent 3,949,471 (issu~~d Aprii 13, 1976, Cawley) comprises a process and a gel cutting device constrdacted and adapted to remove gel like material from a surlEace.
The device has a hollow body, a pickup conduit attached to i:he inlet of the body, a cutter on thf;
inlet end of the pickup conduit. When passed into gel material, the cutter of the pickup conduit cuts the gel and passes the removed material through the pickup conduit into the hollow body. The outlet of the body is connectable to a vacuum source. The removed material can then be recovered.
U.S. Patent 5,5587,062 (issued December 24, 1996, Togawa) is an apparatus for collecting samples by gel electrophoresis, including a base table, a cutting tool or an extractor, a moving mechanism, a pattern detector and a control device, W~ 01/94526 (published December 13, 2001, I~ozulic) is a ge:l cutting and recovering device made up of at least two parts: (1) a hollow member with a distal end terminating in a cutting edge, a proximal end, and a lumen iin between the proximal and distal ends, and (2) a piston. 'The lumen part close to the proximal end has a larger cross section than a lumen part close to the distal end. The piston body fits snugly in the lumen part with the larger cross section and is longer than the length of the lumen part with a smaller cross section and fits within the smaller cross section portion of the hollow tube. This creates reduced pressure when moved towards proximal end of the hollow tube. After cutting a selected portion of the geI mass with the cutting end of the hollow member, and thereby forcing a cut l9ortion of the gel mass into the smaller cross section portion of the lumen, movement of the piston away from the cutting edge reduces the pressure in the smaller cross section of the lumen. This serves to keep the cut gel piece in lumen of the hollow member.
The cut gel can then be ejected by reversing the movement of tlhe piston. In a preferred embodiment, the cut gel portion can be recovered.
CA 2362984 (filed Februaxy 17, 2000, Kevin et al.) is a method and apparatus for the separation and excision of individual protein samples from two-dimensional electrophoresis gels for subsequent analysis of protein content. It addresses the problem of contamination and the need for high throughput by automation with the incorporation of a laboratory robot, a fixed cutting tool arm and tip used with interchangeable or disposable tips. This automated invention would be suited to laboratories handling laxge volumes of samples.
IJ.S. Patent 6,520,036 (issue°,d February 1 ~, x.003, I)weck et al.) uses an external pipetor for excising and ejecting a section of an agarose gel sample from a tray produced by agarose gel electrophoresis. The inner surface ~~f the tip can be scratched, increasing frictional engagement of the cut gel with the inner surface of the tip and rendering the device to work well on some types of gel but not others.
The advantage of the proposed apparatus is that the gel is run normally and the bands are allowed to separate as per usual. fIowever, the piece of gel containing the specimen is then cut and placed in the gel extractor, which uses the running buffer as the f nal elution buffer, so there is no manipulation of solutions. Also, electrophoresis is used to extract the specimen from the cut gel so that all of the specimen ends up in the final solution and is not wasted.
Also, since there is no waiting for drying of beads or continuous stopping and reapplying of the potential, this method is much faster than the other two methods.
This invention combines several steps of separating, removing and recovering desired molecular specimens in a single device that does not require elaborate and expensive construction.
It is not limited by a minimum sample size to be efficient. It does not require separate optical detector to enable the user to determine which part of the gel or which migration band is of interest.
Instead, in this invention, duplicate lanes of the desired sample are run on the gel. The position of the desired sample is visually detected by applying ethidium bromide to one lane of the sample.
This permits positioning and excising duplicate sample in the alternate lane without contamination by a staining agent. In addition, this invention is versatile and can be 'adapted to the type of molecular species that can be handled.
Detailed Description of the Invention The apparatus representing the invention is an electrophoretic apparatus for extracting a molecular specimen from a standard polymer gel. The assumption is made that an initial gel electrophoresis {Figure 6) using a polymer gel carried out in slab form supported by two glass plates has been performed in order to segregate the desired molecular specimen from contaminating species into a band (12, Figure 6). :Following this initial electrophoresis, the uppermost glass plate is removed so that the gel slab is free to be manipulated. Figure 1 shows the gel extraction unit, which is used to excise the desired band from the initial gel electrophoresis. The gel extractor is composed of a sample collecting chamber (1) whose walls are made of a non-conducting material {2), and a gel holding chamber (3). The desired band of the initial gel will then be excised using the cutting edge {4) of the gel holding chamber to ensure proper sizing. This cutting edge ensures that the excised piece of gel containing the desired sample becomes packed into the gel holding chamber. The bottom edge of the cutting edge is tailored to specifically perforate the gel such that the gel piece has the exact dimensions as the gel holding chamber.1-laving the sample collecting chamber sectioned from the gel holding chamber ensures that the gel piece remains in the gel holding chamber.
Figure 2 represents the electrode containing cap that consists of an attractive electrode (5).
The stalk (6) contains a wire that can be attached to a voltage supply unit.
The electrode is capped by a non-conductive or electrical insulating material (7). This electrode is removable, and can be snapped directly onto the sample collecting chamber of the gel extraction unit. The electrode is allowed to contact the polar fluid in. the sample collecting chamber in order to establish an effective electrolytic migration of ions.
Figure 3 shows the repulsive electrode (~) made of conducting material that is attached to a wire (10). The wire is surrounded by an electrical insulating material (9).
The repulsive electrode is removable, and can be used to slide the gel piece from the cutting edge of the gel extraction unit to the segregation point between the gel holding chamber and the sample collecting chamber. An electrolytic buffer is added prior to attachment to a voltage supply in order to set up the appropriate migration path. The dimensions of the repulsive electrode are such that when placed in the gel holding chamber a tight seal is foryraed to prevent the leakage of buffer.
Figure 4 represents the bottcom view of the cap (7) which attaches to the sample collecting chamber. The figure shows the stalk (6) of the attractive electrode that passes through the centre.
The attractive electrode (S) is also shown. Notice that both the sample collecting chamber and the attractive electrodes are cylindrical.
Figure 5 is the bottom view of the adjustable repulsive electrode. This electrode fits tightly into the gel holding chamber to prevent leakage. The piece of gel cut from the initial electrophoresis would lie behind the repulsive electrode and beneath the page.
Figure 6 represents the initial polymer gel (11) that contains the molecular bands of the desired molecular species (12) to be; excised and isolated. The area to be excised is shown by (13).
Figure 7 is the cross-section of the gel being cut out by the gel extraction unit. The cutting edge pierces the gel and the desired area is retained in the distal end of the gel holding chamber.
Figure 8 represents the gel extraction apparatus after the desired molecular band has been excised from the initial polymer gel. The gel extractor is simply pullec~L up and away from the initial gel, and the excised band is removed with it.
Figure 9 shows the complete set up of the gel extraction apparatus. The adjustable, removable repulsive electrode is pushed into the gel holding chamber of the gel extraction unit. As a result, the gel piece is forced all the way back in the geI holding chamber until it contacts the segregation area between the gel holding chamber and the sample collecting chamber. The removable repulsive electrode is removed so that an electrolytic buffer can be inserted. The repulsive electrode is reintroduced into the gel holding chamber; its dimensions are such that a perfect seal is formed between the walls of the gel holding chamber and the electrode. An appropriate fluid, which may consist of a specialized collection or recovery buffer, is put into the sample collecting chamber. The att7~active electrode containing cap is then placed on the sample collecting chamber. The stalks of the repulsive and attractive electrodes are connected to a voltage supply in order to create an electrical potential. Due to this potential, the desired species in the band in the gel piece held by the gel holding chamber migrates out of the polymer gel and into the solution in the sample collecting chamber. Once all of the species has migrated out of the gel piece and into the recovery solution in the sample collecting chamber, the voltage supply is disconnected.
The attractive electrode containing cap is separated and the solution containing the desired species can be removed by pipetting. The species, already dissolved in a recovery fluid, is ready for further experimental manipulations.
It should be understood that the foregoing description is not meant to limit the invention.
Although the utility of the invention has been described with reference to a gel electrophoresis that has been completed initially by another apparatus, this is not intended to limit the scope of the present invention. Furthermore, there are many alternatives and alterations that can be devised by people skilled in the art without departing from the spirit of the invention.
Examples would include isolating proteins from acrylamide gels, varying sizes and shapes for the chamber. The present invention is intended to embrace all such alternatives, modifications and variances which fall into the scope of the appended claims.
In research laboratories, it is often necessary to separate and isolate a particular molecular specimen, such as DNA or protein, from a mixture. This is often done by electrophoresis, which uses an electrical potential to move electrically charged particles through a polymer gel and separate different specimens based on a particular characteristic such as size, charge, or polarity. After separating the molecular specimen in the gel, it is sometimes reeluired to recover a particular sample of interest from the gel Prior devices or methods have addressed the purification, concentration, segregation, or transfer of molecular species from gel media. Some prior devices were developed for specific molecular species (nucleic acids or proteins).
U.S. Patent S,44S,723 (issued August, 1995, Camacho) is a blotting apparatus for transferring electrophoretically separated material from electrophoresis gels in a transfer stack to an immobilizing membrane. To recover the sample, additional steps will be required.
U.S. Patent 5,102,518 (issued April 7, 1992, Doering et al.) is a device for electroeluting and concentrating small amounts of proteins from sample gels for subsequent concentration or processing in a sequencing device with minimized sample handling and minimized disturbance. It elutes desired molecules from gels in a sample holder directly onto a membrane of a centrifuge concentrator or a sequencing membrane.
U.S. Patent 5,217,593 (issued June 8, 1993, I~acConnell) describes a process for purifying DNA and the like. It selectively applies an electrical potential along an inlet path for selectively moving a plasmid into an elution chamber, then along an outlet path into a collection chamber with an elution window at the outlet end.
U.S. Patent 5,384,022 (issued January 24, 1995, Rajasekaran) incorporates a method and apparatus for electrophoretic DNA band isolation and recovery with improved yields of recovered DNA. It includes a second well downstream in the migration path of the molecular specimen. A
cup, having a rim, is inserted into tlhe second well. Together with a membrane also placed in the second well, the molecular specimen is prevented from migrating past the cup.
The molecular specimen migrates into the cup and. is aspirated.
U.S. Patent 6,146,511 (issued November 14, 2000, Slater et al.) is a 2-step electrophoretic method focussing on the purification of a nucleic acid sample by reducing the concentration of contaminants relative to the concentration of desired nucleic acid in the nucleic acid sample.
Several electrophoresis-based prior instruments were designed with specific devices for cutting/ excising and removing/recovering separated specimens from gel media.
U.S. Patent 3,949,471 (issu~~d Aprii 13, 1976, Cawley) comprises a process and a gel cutting device constrdacted and adapted to remove gel like material from a surlEace.
The device has a hollow body, a pickup conduit attached to i:he inlet of the body, a cutter on thf;
inlet end of the pickup conduit. When passed into gel material, the cutter of the pickup conduit cuts the gel and passes the removed material through the pickup conduit into the hollow body. The outlet of the body is connectable to a vacuum source. The removed material can then be recovered.
U.S. Patent 5,5587,062 (issued December 24, 1996, Togawa) is an apparatus for collecting samples by gel electrophoresis, including a base table, a cutting tool or an extractor, a moving mechanism, a pattern detector and a control device, W~ 01/94526 (published December 13, 2001, I~ozulic) is a ge:l cutting and recovering device made up of at least two parts: (1) a hollow member with a distal end terminating in a cutting edge, a proximal end, and a lumen iin between the proximal and distal ends, and (2) a piston. 'The lumen part close to the proximal end has a larger cross section than a lumen part close to the distal end. The piston body fits snugly in the lumen part with the larger cross section and is longer than the length of the lumen part with a smaller cross section and fits within the smaller cross section portion of the hollow tube. This creates reduced pressure when moved towards proximal end of the hollow tube. After cutting a selected portion of the geI mass with the cutting end of the hollow member, and thereby forcing a cut l9ortion of the gel mass into the smaller cross section portion of the lumen, movement of the piston away from the cutting edge reduces the pressure in the smaller cross section of the lumen. This serves to keep the cut gel piece in lumen of the hollow member.
The cut gel can then be ejected by reversing the movement of tlhe piston. In a preferred embodiment, the cut gel portion can be recovered.
CA 2362984 (filed Februaxy 17, 2000, Kevin et al.) is a method and apparatus for the separation and excision of individual protein samples from two-dimensional electrophoresis gels for subsequent analysis of protein content. It addresses the problem of contamination and the need for high throughput by automation with the incorporation of a laboratory robot, a fixed cutting tool arm and tip used with interchangeable or disposable tips. This automated invention would be suited to laboratories handling laxge volumes of samples.
IJ.S. Patent 6,520,036 (issue°,d February 1 ~, x.003, I)weck et al.) uses an external pipetor for excising and ejecting a section of an agarose gel sample from a tray produced by agarose gel electrophoresis. The inner surface ~~f the tip can be scratched, increasing frictional engagement of the cut gel with the inner surface of the tip and rendering the device to work well on some types of gel but not others.
The advantage of the proposed apparatus is that the gel is run normally and the bands are allowed to separate as per usual. fIowever, the piece of gel containing the specimen is then cut and placed in the gel extractor, which uses the running buffer as the f nal elution buffer, so there is no manipulation of solutions. Also, electrophoresis is used to extract the specimen from the cut gel so that all of the specimen ends up in the final solution and is not wasted.
Also, since there is no waiting for drying of beads or continuous stopping and reapplying of the potential, this method is much faster than the other two methods.
This invention combines several steps of separating, removing and recovering desired molecular specimens in a single device that does not require elaborate and expensive construction.
It is not limited by a minimum sample size to be efficient. It does not require separate optical detector to enable the user to determine which part of the gel or which migration band is of interest.
Instead, in this invention, duplicate lanes of the desired sample are run on the gel. The position of the desired sample is visually detected by applying ethidium bromide to one lane of the sample.
This permits positioning and excising duplicate sample in the alternate lane without contamination by a staining agent. In addition, this invention is versatile and can be 'adapted to the type of molecular species that can be handled.
Detailed Description of the Invention The apparatus representing the invention is an electrophoretic apparatus for extracting a molecular specimen from a standard polymer gel. The assumption is made that an initial gel electrophoresis {Figure 6) using a polymer gel carried out in slab form supported by two glass plates has been performed in order to segregate the desired molecular specimen from contaminating species into a band (12, Figure 6). :Following this initial electrophoresis, the uppermost glass plate is removed so that the gel slab is free to be manipulated. Figure 1 shows the gel extraction unit, which is used to excise the desired band from the initial gel electrophoresis. The gel extractor is composed of a sample collecting chamber (1) whose walls are made of a non-conducting material {2), and a gel holding chamber (3). The desired band of the initial gel will then be excised using the cutting edge {4) of the gel holding chamber to ensure proper sizing. This cutting edge ensures that the excised piece of gel containing the desired sample becomes packed into the gel holding chamber. The bottom edge of the cutting edge is tailored to specifically perforate the gel such that the gel piece has the exact dimensions as the gel holding chamber.1-laving the sample collecting chamber sectioned from the gel holding chamber ensures that the gel piece remains in the gel holding chamber.
Figure 2 represents the electrode containing cap that consists of an attractive electrode (5).
The stalk (6) contains a wire that can be attached to a voltage supply unit.
The electrode is capped by a non-conductive or electrical insulating material (7). This electrode is removable, and can be snapped directly onto the sample collecting chamber of the gel extraction unit. The electrode is allowed to contact the polar fluid in. the sample collecting chamber in order to establish an effective electrolytic migration of ions.
Figure 3 shows the repulsive electrode (~) made of conducting material that is attached to a wire (10). The wire is surrounded by an electrical insulating material (9).
The repulsive electrode is removable, and can be used to slide the gel piece from the cutting edge of the gel extraction unit to the segregation point between the gel holding chamber and the sample collecting chamber. An electrolytic buffer is added prior to attachment to a voltage supply in order to set up the appropriate migration path. The dimensions of the repulsive electrode are such that when placed in the gel holding chamber a tight seal is foryraed to prevent the leakage of buffer.
Figure 4 represents the bottcom view of the cap (7) which attaches to the sample collecting chamber. The figure shows the stalk (6) of the attractive electrode that passes through the centre.
The attractive electrode (S) is also shown. Notice that both the sample collecting chamber and the attractive electrodes are cylindrical.
Figure 5 is the bottom view of the adjustable repulsive electrode. This electrode fits tightly into the gel holding chamber to prevent leakage. The piece of gel cut from the initial electrophoresis would lie behind the repulsive electrode and beneath the page.
Figure 6 represents the initial polymer gel (11) that contains the molecular bands of the desired molecular species (12) to be; excised and isolated. The area to be excised is shown by (13).
Figure 7 is the cross-section of the gel being cut out by the gel extraction unit. The cutting edge pierces the gel and the desired area is retained in the distal end of the gel holding chamber.
Figure 8 represents the gel extraction apparatus after the desired molecular band has been excised from the initial polymer gel. The gel extractor is simply pullec~L up and away from the initial gel, and the excised band is removed with it.
Figure 9 shows the complete set up of the gel extraction apparatus. The adjustable, removable repulsive electrode is pushed into the gel holding chamber of the gel extraction unit. As a result, the gel piece is forced all the way back in the geI holding chamber until it contacts the segregation area between the gel holding chamber and the sample collecting chamber. The removable repulsive electrode is removed so that an electrolytic buffer can be inserted. The repulsive electrode is reintroduced into the gel holding chamber; its dimensions are such that a perfect seal is formed between the walls of the gel holding chamber and the electrode. An appropriate fluid, which may consist of a specialized collection or recovery buffer, is put into the sample collecting chamber. The att7~active electrode containing cap is then placed on the sample collecting chamber. The stalks of the repulsive and attractive electrodes are connected to a voltage supply in order to create an electrical potential. Due to this potential, the desired species in the band in the gel piece held by the gel holding chamber migrates out of the polymer gel and into the solution in the sample collecting chamber. Once all of the species has migrated out of the gel piece and into the recovery solution in the sample collecting chamber, the voltage supply is disconnected.
The attractive electrode containing cap is separated and the solution containing the desired species can be removed by pipetting. The species, already dissolved in a recovery fluid, is ready for further experimental manipulations.
It should be understood that the foregoing description is not meant to limit the invention.
Although the utility of the invention has been described with reference to a gel electrophoresis that has been completed initially by another apparatus, this is not intended to limit the scope of the present invention. Furthermore, there are many alternatives and alterations that can be devised by people skilled in the art without departing from the spirit of the invention.
Examples would include isolating proteins from acrylamide gels, varying sizes and shapes for the chamber. The present invention is intended to embrace all such alternatives, modifications and variances which fall into the scope of the appended claims.
Claims (13)
1 - An apparatus used to isolate molecular species from an electrophoretic polymer gel by cutting a specified part of said gel and using electrophoresis to cause said molecular species to migrate out of said specified part of said gel. Said apparatus is comprised of a cutting edge to cut out said specified part of said gel from said gel, a gel holding chamber to hold said specified part of said gel, a sample collecting chamber to recover said molecular species into a recovery solution, a repulsive electrode, and an attractive electrode.
2 - An apparatus as in claim 1 where said repulsive electrode may be used to alter the volume of said gel holding chamber by sliding along the length of said gel holding chamber.
3 - An apparatus as in claim 1 wherein said attractive electrode is contained in a cap which can be used to seal said sample collection chamber.
4 - An apparatus as in claim 1 wherein the said attractive electrode is covered by a membrane which excludes the said molecular species.
- An apparatus as in claim 1 further comprising a voltage supply, where said voltage supply may comprise a battery.
6 - An apparatus as in claim 1 composed of but not limited to a non-conducting material.
7 - An apparatus as in claim 1 wherein said gel holding chamber has dimensions larger than those of said sample collection chamber.
8 - An apparatus as in claim 1 wherein said gel holding chamber is coupled to said sample collecting chamber.
9 - An apparatus as in claim 8 wherein said gel holding chamber is able to contain electrolytic fluids.
10-An apparatus as in claim 8 wherein said sample collecting chamber is able to contain electrolytic or non-electrolytic solutions.
11-An apparatus as in claim 1 wherein said gel holding chamber has a bottom opening modified to receive excised portion of said gel.
12-An apparatus as in claim 1 wherein said cutting edge is coupled to, but not limited to, said gel holding chamber or said repulsive electrode.
13- The said cutting edge as in claim 12 wherein edge is specifically adapted to excise desired area of gel by compressing against said gel.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2427488 CA2427488A1 (en) | 2003-05-08 | 2003-05-08 | Gel extraction apparatus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2427488 CA2427488A1 (en) | 2003-05-08 | 2003-05-08 | Gel extraction apparatus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2427488A1 true CA2427488A1 (en) | 2004-11-08 |
Family
ID=33426193
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2427488 Abandoned CA2427488A1 (en) | 2003-05-08 | 2003-05-08 | Gel extraction apparatus |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2427488A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114277027A (en) * | 2021-12-23 | 2022-04-05 | 深圳大学 | Tool for recovering electrophoresis separated substances and use method thereof |
-
2003
- 2003-05-08 CA CA 2427488 patent/CA2427488A1/en not_active Abandoned
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114277027A (en) * | 2021-12-23 | 2022-04-05 | 深圳大学 | Tool for recovering electrophoresis separated substances and use method thereof |
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