WO2020146565A1 - Cassette moulée à gel et système de moulage à la main - Google Patents

Cassette moulée à gel et système de moulage à la main Download PDF

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Publication number
WO2020146565A1
WO2020146565A1 PCT/US2020/012829 US2020012829W WO2020146565A1 WO 2020146565 A1 WO2020146565 A1 WO 2020146565A1 US 2020012829 W US2020012829 W US 2020012829W WO 2020146565 A1 WO2020146565 A1 WO 2020146565A1
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WO
WIPO (PCT)
Prior art keywords
frame
cassette
cassette frame
cassete
closed position
Prior art date
Application number
PCT/US2020/012829
Other languages
English (en)
Inventor
Kurt Greenizen
Christopher Scott
Paul Sydlowski
Kevin Wu
Ryan Amara
Ryan Manning
Sara Gutierrez
John Doyle
Original Assignee
Emd Millipore Corporation
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Emd Millipore Corporation filed Critical Emd Millipore Corporation
Publication of WO2020146565A1 publication Critical patent/WO2020146565A1/fr

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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

Definitions

  • Typical blocking agents include casein, bovine serum albumin (BSA), non-fat dry milk (generally about 1-5%) in a Tris buffer saline solution with TWEEN® surfactant (TBS-T solution), or phosphate buffer saline solution with TWEEN® surfactant (PBS-T solution).
  • BSA bovine serum albumin
  • TBS-T solution Tris buffer saline solution with TWEEN® surfactant
  • PBS-T solution phosphate buffer saline solution with TWEEN® surfactant
  • a detection reagent generally a chromogenic, chemiluminescent, fluorescent, radiological, or streptavidin- labeled material, is applied which either binds to, or is a substrate of the enzyme- conjugate.
  • the appropriate detection device is used to determine the presence, absence, position, quantity, etc. of the biological entity.
  • the first step in the casting process begins by injecting (usually by pipette) liquid acrylamide resolving gel into the cassette.
  • this requires the user to inject into what is a narrow opening at the top of each individual cassette.
  • Multicast systems may allow the users to flood the entire stack simultaneously but produce an excess of acrylamide waste which must be cleaned after the process is completed.
  • Visibility into the cassette is important to achieve the desired height of the resolving gel which is advantageous in the single cassette systems but not achievable beyond the first couple of layers in a multicast system.
  • the second step in the casting process involves injecting the acrylamide stacking gel into each cassette, again usually by pipette. This also requires the user to inject into the narrow opening at the top of each individual cassette.
  • the user inserts a sample well comb matched to the gel thickness.
  • the combs are constructed with a number of teeth to form wells, the number of which is based on sample size and desired well volumes (common configurations are 10, 12, and 15 wells). While inserting the comb, it is important for it to be centered from side to side with respect to the cassette and remain secure both during and after the stacking gel cures. It is also important that the comb can be removed (for electrophoresis sample loading) without disruption of the wells.
  • a gel casting device including a cassette frame comprising first and second arms or members pivotally connected to one another.
  • One or more hinges may be provided to allow the first member to swing or pivot relative to the second member (or vice versa) between a cassette frame open position and a cassette frame closed position.
  • the first and second members each include a substrate to maintain a gel interface, preferably a glass plate substrate.
  • one of the first and second members may have an integral molded seal, and the other of the first and second members may have a corresponding rigid element that cooperates with the integral molded seal to provide a seal interface when the first and second members are in the closed position.
  • At least one of the first and second members has an expanded access region to allow for high volume fluid loading.
  • openings such as elongated slots are provided in the first and second arms or members to allow for electrophoresis buffer access and the like to the region defined between the substrate when the first and second members are in the closed position.
  • a multi-well comb may have flexible alignment tabs which interface with one or more hinges on the cassette frame and aid in the proper positioning of the comb within the device. This ensures consistent well formation for sample introduction into the cassette.
  • the comb may also have ejection tabs to assist the user in properly removing the comb from the device.
  • an assembly comprises one or more cassette frames, and a base for holding one or more cassette frames, the base having pivot arms that engage and compress the one or more cassette frames in a fluid-tight manner.
  • a cassette frame for casting an electrophoretic separation medium comprising a first arm including a first substrate having a first substrate inner major surface; a second arm pivotal with respect to the first arm between a cassette frame open position and a cassette frame closed position, the second arm including a second substrate having a second substrate inner major surface; wherein when the cassette frame is in its closed position, the first substrate inner major surface and the second substrate inner major surface face each other and define between them an electrophoretic separation medium receiving region.
  • the first and second substrates comprise respective glass plates.
  • the first and second arms each comprise an opening providing access to the electrophoretic separation medium receiving region when the cassette frame is in the closed position.
  • the first and second arms each comprise more than one opening providing access to an electrophoretic separation medium receiving region.
  • the first and second members are secured in the closed position with slidably attached side arms.
  • the first and second arms are secured in the closed position with side arms attached to the cassette frame with a clasp, a hinge slide, or a snap lock.
  • the cassette frame includes a pair of spaced hinges enabling the second arm to pivot with respect to the first arm.
  • the first arm comprises an angled wall defining a fluid reservoir when the cassette frame is in its closed position.
  • the pins of the spaced hinges have flat surfaces opposite of one another to allow separation of the first and second arms when the cassette frame is in said open position.
  • the first and second arms are separable when the cassette frame is opened 270° relative to said closed position.
  • an assembly for casting an electrophoresis separation medium comprising at least one cassette frame having a cavity for containing an electrophoretic separation medium; a base having a cassette frame receiving region for holding the at least one cassette frame; a pair of clamping arms pivotally connected to the base, each clamping arm being pivotable between an open position where a clamping force is not applied to the at least one cassette frame, and a closed position where a clamping force is applied to the at least one cassette.
  • the base is configured to hold a plurality of cassette frames in compressed relation.
  • the cassette receiving region holds the cassette frame using walls hingedly attached to the base. In certain embodiments, the walls are engaged to hold the cassette frame by contact of the cassette frame to the base.
  • an assembly for casting an electrophoresis separation medium comprising a first arm including a first substrate having a first substrate inner major surface; a second arm pivotal with respect to the first arm between a cassette frame open position and a cassette frame closed position, the second arm including a second substrate having a second substrate inner major surface; a comb having a plurality of teeth; wherein when the cassette frame is in the closed position, the first substrate inner major surface and the second substrate inner major surface face each other and define between them an electrophoretic separation medium receiving region, and wherein the comb is insertable into the cassette frame when in the cassette frame closed position such that the plurality of teeth of the comb extend into electrophoretic separation medium receiving region.
  • the cassette frame comprises a pair of spaced hinges enabling the first arm to pivot with respect to the second arm, and the comb comprises a pair of spaced alignment tabs, each of which interfaces with a respective one of the spaced hinges.
  • an assembly for casting an electrophoresis separation medium comprising: at least one cassette frame having a cavity for containing an electrophoretic separation medium; a base having a cassette frame receiving region for holding said at least one cassette frame, the cassette receiving region comprising a pair of walls hingedly attached to the base, wherein the pair of walls are engaged to hold said at least one cassette frame by contact of the cassette frame receiving region with said at least one cassette frame; a pair of clamping arms pivotally connected to said base, each of said clamping arms being pivotable between an open position where a clamping force is not applied to said at least one cassette frame, and a closed position where a clamping force is applied to said at least one cassette.
  • Various embodiments are disclosed herein of a method for casting an electrophoresis separating medium using the assembly disclosed herein, the method comprising: sliding side arms onto said at least one cassette frame to secure said at least one cassette frame in the closed position; contacting said at least one cassette frame with the base having the pair of clamping arms in the open position; engaging the pair of walls hingedly attached to said base to hold said at least one cassette frame; pivoting the pair of clamping arms connected to said base into the closed position; applying a gel solution to the fluid reservoir through the opening of said at least one cassette frame, thereby casting an electrophoresis separating medium.
  • FIG. 1A is a perspective view of a cassette frame in accordance with certain embodiments
  • FIG. IB is a perspective view of a cassette frame in accordance with certain embodiments
  • FIG. 1C is a perspective view of a cassette frame in accordance with certain embodiments.
  • FIG. ID is a perspective view of a cassette frame in accordance with certain embodiments.
  • FIG. 2A is a perspective view, in cross-section, of a cassette frame in accordance with certain embodiments
  • FIG. 2B is a perspective view, in cross-section, of a cassette frame in accordance with certain embodiments
  • FIG. 3 is a perspective view, in cross-section, of a portion of a cassette frame in accordance with certain embodiments
  • FIG. 4A is a perspective view of a comb in accordance with certain embodiments.
  • FIG. 4B is a perspective view of a comb in accordance with certain embodiments.
  • FIG. 5 is a perspective view in cross-section of the comb of FIG. 4 shown inserted in a cassete frame in accordance with certain embodiments;
  • FIG. 6 is a perspective view of a comb shown inserted in a cassette frame in accordance with an alternative embodiment
  • FIG. 7A is a perspective view of a cassette frame positioned in a cassete clamp stand in accordance with certain embodiments
  • FIG. 7B perspective view of a cassete frame positioned in a cassete clamp stand in accordance with certain embodiments
  • FIG. 8A is a perspective view of a cassete assembly including multiple cassete frames positioned and clamped in a clamp stand in accordance with certain embodiments;
  • FIG. 8B is a perspective view of a cassete assembly including multiple cassete frames positioned and clamped in a clamp stand in accordance with certain embodiments;
  • FIG. 9A is a partial perspective view, in cross-section, of a cassete assembly including multiple cassete frames positioned in a clamp stand in accordance with certain embodiments;
  • FIG. 9B is a partial perspective view, in cross-section, of a cassette assembly including multiple cassette frames positioned in a clamp stand in accordance with certain embodiments;
  • FIG. 9C is a partial perspective view, in cross-section, of a cassette assembly including multiple cassette frames positioned in a clamp stand in accordance with certain embodiments;
  • FIG. 10 is a perspective view of a clamp stand in accordance with an alternative embodiment
  • FIG. 11 is a perspective view of the clamp stand of FIG. 10 with cassette frames mounted therein in accordance with certain embodiments.
  • FIG. 12A is a perspective view of a gel scraper in accordance with certain embodiments.
  • FIG. 12B is a diagrammatic view showing the gel scraper of FIG. 12A opening a cassette frame in accordance with certain embodiments
  • FIG. 12C is a diagrammatic view showing the gel scraper of FIG. 12A trimming gel in a cassette frame in accordance with certain embodiments;
  • FIG. 12D is a diagrammatic view showing the gel scraper of FIG. 12A removing gel from a cassette frame in accordance with certain embodiments.
  • FIG. 13 is a perspective view of a cassette assembly including multiple cassette frames having fluid level indicators positioned and clamped in a clamp stand in accordance with certain embodiments.
  • the cassette frame 10 may be constructed of one or more molded pieces chemically compatible with gel casting solutions, such as polycarbonate, and may include a first arm or member 12 having a top side 12A and a bottom side (not shown).
  • the member 12 is generally planar and rectangular. It may have a free end 13 A and a pivotal end 13B spaced from the free end 13A.
  • the pivotal end 13B may include spaced ears 14A, 14B that together with respective pins 26, cooperate to form a pair of spaced hinges 35 A, 35B.
  • the pins 26 of the pair of spaced hinges 35A, 35B have flat surfaces 26A opposite of one another to allow separation of the members 12 and 22 of the cassette frame 10 when in the open position.
  • the members 12 and 22 are separable when the cassette frame 10 is opened 270° relative to the closed position. Separation improves the ability of a user to sufficiently clean the cassette frame 10 between uses.
  • One or more slots 8 A may be provided in the member 12 such as to provide access to the interior cavity or chamber of the cassette frame 10 such as for the introduction of electrophoresis buffer and the like when the frame is in its closed condition.
  • an elongated slot 8A is provided near the free end 13 A as shown.
  • the elongated slot 8A is provided near the pivotal end 13B as shown in FIG. IB, FIG. 1C, and FIG. ID.
  • An access region or reservoir 16 may be provided near the pivotal end 13B, and the access region or reservoir 16 may be configured to hold a predetermined volume of fluid so that the fluid may be loaded in the access region 16 to then drain into the device.
  • the access region or reservoir 16 is near the free end 13 A, as shown in FIG. 1B-1D.
  • the access region 16 may include an angled wall 16A offset from the surface of the member 12 as shown.
  • the cassette frame 10 has more than one access region or reservoir 16 as shown in FIG. ID.
  • a substrate preferably a glass plate 20
  • the glass plate 20 is preferably transparent, generally flat and rectangular, and includes a first or inside major surface opposite a second or outside major surface.
  • the cassette frame 10 is secured in the closed condition with side arms 64 slidably attached to the cassette frame 10 with a grove mechanism, as shown in FIG. 1C and FIG. ID. In certain embodiments, the cassette frame 10 is secured with one side arm 64.
  • the side arms 64 attach to the cassette frame 10 using other mechanical attachment, such as a snap lock, a hinge pin, a clasp, and a keyed slot allowing a side arm 64 to stay with one side of the cassette frame.
  • the cassette frame 10 also may include a second arm or member 22 having a top side 22A and a bottom side (not seen).
  • the member 22 may be generally planar and rectangular. It may have a free end 23A and a pivotal end 23B spaced from the free end 23 A.
  • the pivotal end 23B may include spaced ears 24A, 24B that together with the respective pins 26, cooperate to form a pair of spaced hinges 35 A, 35B.
  • One or more slots may be provided in the member 22 to provide access to the interior cavity or chamber of the cassette frame 10 such as for the introduction of electrophoresis buffer and the like.
  • an elongated slot 8B is provided near the pivotal end 23B.
  • the elongated slot 8B is provided near the free end 23 A as shown in FIG. IB.
  • a substrate preferably a glass plate 30, may be affixed to the member 22 as best seen in FIG. 2A and 2B.
  • the glass plate 30 is preferably transparent, generally flat and rectangular, and includes a first or inside major surface opposite a second or outside major surface.
  • the first member 12 and the second member 22 are of the same thicknesses and dimensions.
  • the glass plates 20 and 30 are of the same thicknesses and dimensions.
  • the glass plates 20 and 30 may have different dimensions and/or thicknesses to improve sealing between the comb 40 and the glass plates 20 and 30.
  • the bottom side of member 12 may include a cut-out 27 which receives a seal or gasket, such as an elastomeric seal 28.
  • the top side of the member 22 includes a rigid protrusion 29 that extends upwardly from the generally planar surface of the member 22 and cooperates with the elastomeric seal 28 to provide a seal interface when the members 12 and 22 are in the closed position.
  • side arms 64 are slidably attached to the cassette frame 10 with a grove mechanism to provide a seal interface when the members 12 and 22 are in the closed position.
  • the grove mechanism may be a tongue and grove joint, such that the side arms 64 may be slid on the cassette frame 10 at the free end 13A and/or 23A.
  • attachment of the side arms 64 to the cassette frame 10 reduces the risk of delamination of the gel to be cast in the embodiments disclosed herein.
  • the members 12 and 22 are hingedly or pivotally connected and define respective halves of a clam-shell cassette frame assembly.
  • the cassette frame 10 may be opened (e.g., FIG. 9C) and closed (e.g., FIG. 9A and FIG. 9B) without disruption of glass plates 20 and 30 and the electrophoretic separation medium receiving region, cavity, or chamber 70 (FIG. 9A, FIG. 9B, and FIG. 9C) defined between them.
  • the members 12 and 22 are positioned in overlying relationship defining between them the electrophoretic separation medium receiving region, cavity, or chamber 70.
  • the glass plates 20, 30 are each affixed to a respective member 12 and 22 by any suitable means, such as bonding with an adhesive such as an epoxy, a cyanoacrylate, or UV curable adhesive, or may be attached by over-molding.
  • the thickness of glass plate 20 is smaller than the thickness of the member 12, and the thickness of the glass plate 30 is smaller than the thickness of the member 22.
  • the member 12 provides a border or frame around the perimeter of the glass plate 20, and the member 22 provides a border or frame around the perimeter of the glass plate 30.
  • Optimal electrophoresis separation medium interface is thus provided.
  • the respective inside major surfaces of the glass plates 20 and 30 face each other and do not contact one another; there is a gap between them defining at least in part the electrophoretic separation medium receiving region, cavity or chamber 70.
  • the cassette frame 10 can be configured to achieve a desired electrophoretic separation medium receiving region 70 thickness, and thus ultimately an electrophoretic separation medium (e.g., a gel) thickness.
  • Suitable electrophoretic separation medium thicknesses include 0.75mm, 1.0mm and 1.5 mm, etc., depending at least in part of the sample volume required for the electrophoresis process.
  • FIG. 4A, FIG. 4B, and FIG. 5 there is shown a comb 40 capable of operably associating with a cassette frame 10 for forming sample wells in the electrophoretic separation medium in accordance with certain embodiments.
  • the comb 40 includes a main body region 41 and a plurality of spaced fingers or teeth 42, namely, teeth 42a, 42b, 42c, 42n extending from the main body region 41.
  • teeth 42a, 42b, 42c, 42n there are ten teeth 42 in the embodiment shown, although those skilled in the art will appreciate that more or fewer teeth 42 may be suitable, depending upon the number of sample wells desired to be formed in the electrophoretic separation medium.
  • each of the teeth 42 extends from the main body region 41 in the same direction and to the same extent, is of the same size and shape (generally rectangular) and terminates in a free end.
  • Proper positioning of the comb 40 in the hinged end 10A of the cassette frame 10 results in the teeth 42 extending into the electrophoretic separation medium receiving region 70 and enables the formation of tooth-shaped wells in the electrophoretic separation medium or gel to form well shapes matching the shape of the teeth 42. That is, upon polymerization of the medium, the teeth 42 serve to occupy regions that the medium would otherwise occupy, so that upon polymerization of the medium and subsequent removal of the comb 40, regions devoid of polymerized medium remain and define the wells for introduction of sample to be subjected to electrophoresis.
  • Suitable materials of construction for the comb include those compatible with gel casting solutions such as polycarbonate.
  • user interface region 43 extending from the main body region 41 in a direction opposite to the teeth 42 is user interface region 43, such as an integral arc-shaped member that facilitates grasping of the comb 40 by the user for insertion and removal of the comb 40 from the cassette frame 10.
  • the comb 40 may include spaced flexible alignment tabs 45A, 45B which interface with the cassette frame hinges 35A, 35B and assist in centering the comb 40 within the electrophoretic separation medium receiving region defined between the members 12 and 22 when in the closed position to ensure consistent well positioning for sample introduction.
  • each of the flexible alignment tabs 45 A, 45B may include a recessed surface that receives a corresponding respective dome-shaped protrusion 46A, 46B extending radially from each hinge 35A, 35B, as best seen in FIG. 5.
  • FIG. 5 As seen in FIG.
  • spaced ejection tabs 47 A, 47B may be provided on the comb 40 to assist in removing the comb from the cassette frame 10 upon completion of well formation.
  • the ejection tabs 47 A, 47B may include respective pointed projections 48A, 48B that interface with the top surface of respective hinges 35A, 35B.
  • the pointed projections 48A, 48B engage against the hinges 35 A, 35B and provide a mechanical advantage to the user and assist in ejecting the comb 40 from the cassette frame 10 in a direction perpendicular to the formed wells.
  • the comb 40 does not include ejection tabs 47 A, 47B.
  • the assembly may include a cassette clamp stand 50, as shown in FIGS. 7A, FIG. 7B, FIG. 8A, FIG. 8B, and FIG. 9A-9C.
  • the cassette clamp stand 50 may serve multiple functions, including eliminating the need for aligning the cassette frames 10 during assembly, as the cassette frames 10 self-align when positioned in the stand 50); providing compressive force to seal the cassette frame(s) 10, thereby reducing the likelihood of external leaking of electrophoretic separation medium forming components such as acrylamide; providing clear visibility to the cassettes during processing; and simplifying cleaning of the casting apparatus.
  • Suitable materials of construction for the stand 50 include thermoplastics such as polyoxymethylene (e.g., DELRIN ® ), and other materials with equivalent strength durability and a low coefficient of friction relative to the material used for the cassette frame 10.
  • the cassette clamp stand 50 includes a base 51 having a cassette receiving region 49 defined by a front wall 52A, rear wall 52B and opposite side walls 52C and 52D extending vertically from the base 51.
  • the distance between the front and rear walls 52A, 52B may be chosen to receive and hold one or two or more cassette frames 10 in compressed relation when the frames are in their closed positions.
  • the cassette receiving region 49 is configured to receive two cassette frames 10 in front-to-back compressed relation to enable vertical casting of an electrophoretic separation medium in a liquid leak-tight manner, and/or vertical electrophoresis.
  • the front wall 52A and rear wall 52B are hingedly or pivotally attached to base as shown, for example, in FIG. 7B, using hinges 65.
  • the front wall 52A and rear wall 52B may be engaged to hold the cassette frame 10 upon insertion of a cassette frame 10 into the cassette receiving region 49 and to further assist in providing a seal interface when the members 12 and 22 are in the closed position.
  • This design limits the needed insertion/loading force when assembling the cassette with the base to form the gel electrophoresis medium casting assembly.
  • Other compression seal geometries may be suitable to hold fewer or more cassette frames 10 in compressed relation.
  • the clamping stand 50 may include a pair of vertical clamping arms 55 A, 55B, each pivotally connected to the base 51 by respective rotary hinges 56A, 56B.
  • Each clamping arm 55A, 55B may be pivoted between an open position (e.g., (FIG. 7A and FIG. 7B) where it does not contact and clamp a cassette frame 10, and a closed position (FIG. 8A and 8B) where it contacts and clamps one or more cassette frames 10 with a suitable clamping force.
  • each clamping arm 55A, 55B includes a pair of spaced clamping walls 57A, 57B that are configured and positioned to receive side edges of the cassette frames 10 when the clamping arms 55A, 55B are in the closed position.
  • the compressive clamping force applied to the cassette frames 10 by the stand 50 and the clamping arms 55 A, 55B seals the slots in the cassette frame(s) 10 to prevent leaking.
  • the clamping arms 55A, 55B include top clamping member 66 as shown in FIG. 7B and FIG. 8B. The top clamping member 66 may further assist in providing a seal interface when the members 12 and 22 are in the closed position and assist in insuring the cassette frame 10 seats into the cassette receiving region 49.
  • One or more of the clamping arms 55 A, 55B may have visual fill indicators 90 (FIG. 13) affixed to their outer surfaces to provide the user with a visual indication of the fluid level in the cassette frame 10.
  • the indicators may be integral or an add-on such as a sticker adhered to the clamping arm or arms 55 A, 55B.
  • the base 51 may include a plurality of cassette receiving regions 49 (four shown, although fewer or more may be present) that are staggered or offset from one another in order to facilitate visibility into each of the cassette frames 10 clamped therein.
  • FIGS. 12 A, 12B, 12C and 12D illustrate a multi-functional gel scraper 60 in accordance with certain embodiments.
  • the gel scraper 60 may be formed in a wedge shape, with a main body region 61 tapering to anarrow free end 62 as seen in FIG. 12A.
  • a user interface region 63 such as an integral arc-shaped member that facilitates grasping of the gel scraper 60 by the user, may be included.
  • the wedge shape may assist the user in prying open a cassette frame 10, such as for gel removal, when in its closed position, as shown in FIG. 12B.
  • the free end 62 may also serve as a cutting tool for trimming the gel prior to removal from the cassette frame 10 as shown in FIG.
  • the width of the gel scraper 60 and particularly, the width of the free end 62, is configured to match the width of the gel formed on the substrate 20 or 30.
  • Suitable materials of constructions for the gel scraper 60 include polymers with suitable strength and durability such as polycarbonate, ABS, polysulfone and styrene.
  • the cassette clamp stand 50 is placed on a level surface with both vertical clamping arms in the open position.
  • the two cassette frames 10 are placed in back-to-back relation, with the reservoirs 16 facing towards the outside or away from each other, and are inserted into the cassette receiving region of the stand 50. If only one gel is being cast, a second cassette frame may be used as a blank.
  • the vertical clamping arms are then pivoted to the closed or clamping position to seal the cassette frames.
  • a comb may be inserted in a cassette frame by orienting marked side toward the wedge, making sure the alignment tabs properly engage with the cassette frame. A line then may be marked to indicate the desired level of the resolving gel (typically 0.5-1 cm below the teeth of the comb), and then the comb may be removed.
  • the assembly is now ready for the standard protocol for preparing and loading the resolving gel.
  • the system typically requires about 6mL of resolving gel solution per cassette (includes a small amount of excess for monitoring the polymerization progression).
  • a 10% ammonium persulfate (APS) solution in D.I. water is prepared (i.e. lOOmg APS is dissolved in lmL D.I. water. *Note: For best performance prepare fresh APS solution. In an appropriately sized container, acrylamide/bis acrylamide, 1.5M Tris-HCl pH 8.8, D.I. water, 10% sodium dodecyl sulfate (SDS) and 10% APS are combined according to the desired formulation. Immediately before casting, the required amount of TEMED is added and the components are gently mixed. The resolving gel solution is immediately transferred to the casting cassette using a 5 or 10 mL serological pipette.
  • SDS sodium dodecyl sulfate
  • the resolving gel is added to the level previously established followed by an overlay of ethanol or isopropyl alcohol ( ⁇ 0.5-lmL). Polymerization may be monitored using left-over casting solution. Depending on the formulation used, polymerization times will vary (typically 10-30 minutes).
  • the overlay solution may be removed by pouring it off or absorbing it with blotting paper.
  • the alcohol may be completely removed by rinsing with D.I. water, and residual water is removed by absorbing with blotting paper.
  • the stacking gel is then allowed to polymerize (typically 10-30min), and polymerization is verified by monitoring left-over casting solution.
  • polymerize typically 10-30min
  • both vertical clamping arms of the clamp stand are opened to remove casting cassettes. Gels can be used immediately or may be stored at 4°C until use by wrapping in paper towels soaked with D.I. water or running buffer.

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Abstract

Les modes de réalisation de la présente invention concernent un dispositif et un ensemble utiles pour le moulage à la main d'un milieu de séparation électrophorétique. Le dispositif comprend un cadre de cassette 10 comprenant des premier et second bras ou des éléments 12,22 reliés de façon pivotante l'un à l'autre. Une ou plusieurs charnières 35A, 35B peuvent être prévues pour permettre au premier élément d'osciller ou de pivoter par rapport au second élément 12, 22 (ou inversement) entre une position ouverte de cadre de cassette et une position fermée de cadre de cassette. Les premier et second éléments 12, 22 comprennent chacun un substrat 20, 30 pour maintenir une interface de gel, de préférence un substrat de plaque de verre. L'ensemble comprend une base et des bras de serrage qui coopèrent pour maintenir un ou plusieurs cadres de cassette en relation comprimée.
PCT/US2020/012829 2019-01-09 2020-01-09 Cassette moulée à gel et système de moulage à la main WO2020146565A1 (fr)

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US201962790236P 2019-01-09 2019-01-09
US62/790,236 2019-01-09

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EP4006536A1 (fr) * 2020-11-27 2022-06-01 Simo Abdessamad Baallal Jacobsen Ensemble d'electrophorese et methodes

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EP0809104A1 (fr) * 1996-05-20 1997-11-26 Bio-Rad Laboratories, Inc. Cellule électrophorétique pour gels en plaque à assemblage rapide
WO2001081910A1 (fr) * 2000-04-24 2001-11-01 Bio-Rad Laboratories, Inc. Pince de cellule compacte destinee a un ensemble a parois de gel en plaquette
US20020025278A1 (en) * 2001-01-09 2002-02-28 Gel Manipulation Apparatus Gel manipulation apparatus
US20020144879A1 (en) * 2001-04-04 2002-10-10 Anderson N. Leigh Method and apparatus for relieving stress in an electrophoresis gel slab

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