Patent Document

FIELD OF THE INVENTION 
   The present invention relates to a device and method for providing separation media for use in a horizontal electrophoresis system. 
   BACKGROUND OF THE INVENTION 
   Horizontal electrophoresis has been an enduringly popular choice for separation of nucleic acids and proteins and offers several advantages over vertical electrophoresis. Generally, horizontal gels used as separation media are easier to cast than vertical gels. Further, thicker gels can be provided in a horizontal format than in a vertical system, and a lower concentration of separation medium, such as agarose, can be used in horizontal gels than in vertical gels allowing for better separation of high molecular weight molecules. Also, in a horizontal format, sample wells may be provided within the interior of the gel as well as at the top edge, increasing the number of samples that can be loaded per gel. 
   The typical system for casting horizontal gels utilizes a gel-casting tray with a flat bottom surface, two opposing parallel sides and two open ends that must be blocked at set points to retain the separation medium until the medium solidifies, resulting in a gel of limited dimensions. The open ends are usually sealed with an adhesive tape or sealed by the placement of casting gates in pre-cut grooves in the sidewalls and bottom surface of the tray. Whatever method is used, the gel is formed only in specific, predetermined lengths. 
   Several horizontal gel systems, in particular, catalog numbers HG-12, HLB-12, HG-20 and MG-10, available through Tyler Research Corporation, Alberta, Canada, utilize an adjustable gate-placement design. The design allows a casting gate to be placed into one of three placement grooves at pre-set lengths. Although the design increases the flexibility of a system in that it provides for some choice of length to be made, it is still limited to pre-determined lengths. Thus, there is a need for a horizontal gel-casting device that permits the selection of length from along a continuum of lengths. 
   A further disadvantage of systems with pre-determined gate settings is that each setting requires a groove cut in the sidewalls and the otherwise smooth bottom surface of the casting tray for gate placement. Because the grooves are machined for a tight fit with the casting gate, additional grooves add to the cost of manufacturing. Moreover, when a relatively longer gel is poured, the grooves cut in the bottom surface of the casting tray cause variation in gel thickness leading to possible distortion of the separation pattern in the gel. 
   Another limitation associated with using trays with only preset gel dimensions is that the preparation of a gradient gel, in which the concentration of agarose or other ingredients is varied from one end of the gel to the other, is not possible using existing casting tray systems with placement of casting gates only at the ends of the trays, and is not practical using systems with only a few pre-determined gate settings along the length of the tray. Gradient gels are advantageous when separating specimens containing macromolecules over a wide range of sizes. There is a need for a horizontal gel casting system that permits incremental increases, as desired, in the concentration of the separation medium along the length of the gel. 
   SUMMARY OF THE INVENTION 
   The present invention relates, in one aspect, to a device for providing horizontal gels wherein the length of the separation medium is selected from a continuum of possible lengths. Such a feature is beneficial in terms of more efficient use of materials, thus decreasing material costs, and in terms of more effective separation length in accordance with the samples to be analyzed. In another aspect, the invention relates to a method of using the device to produce gels of either a single concentration of separation medium or a gradient of concentrations of separation medium. 
   In its preferred form, the device comprises a casting tray with two opposing parallel sidewalls and two open ends wherein one end is sealed at any point along the length of the tray by a slidable block that acts as a dam to prevent fluid leakage. 
   Various other features, objects and advantages of the invention will be made apparent from the following description taken together with the drawings. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The drawings illustrate the best mode presently contemplated of carrying out the invention. 
     In the drawings: 
       FIG. 1  is a perspective view of a conventional casting tray showing two opposing parallel sides and two open ends. 
       FIG. 2  is a perspective view of a casting tray showing one end sealed with a conventional casting gate and the other end sealed with a sliding block in accordance with a preferred embodiment of the invention. 
       FIG. 3  is a perspective view of a conventional comb used to form sample wells in the gel in accordance with methods of using the invention. 
       FIG. 4  is a perspective view of an empty casting tray sealed at each end showing the comb for forming sample wells in place in one set of placement notches. 
       FIG. 5  is a perspective view of a casting tray filled with solidified separation medium and with the slidable block still in place. 
       FIG. 6  shows one method of using the invention to form a step gradient gel by placing at least two slidable blocks in the casting tray at spaced intervals. 
       FIG. 7  shows the sections of solidified separation medium with voids in between the sections where the blocks had been placed. 
   

   DETAILED DESCRIPTION OF THE INVENTION 
     FIG. 1  is a perspective view of a conventional casting tray  10 . 
   Referring to  FIG. 1 , the casting tray  10  has a generally flat inner surface  12 , two opposing parallel sidewalls  14  and two open ends  16 . The casting tray  10  is preferably made of transparent plastic, such as polymethylmethacrylate or polymethylpentene, molded or machined. Alternatively, the casting tray  10  may be made of any material that would retain a flat surface  12  after repeated use, especially after exposure to molten agarose. The sidewalls  14  have one or more pairs of notches  18 , molded or machined, for positioning one or more combs  20 , shown in  FIG. 3 , for forming sample wells  36 , as shown in  FIG. 7 . A groove  22  running across the surface  12  and into each opposing sidewall  14  serves for placement of a casting gate  24 , as shown in  FIG. 2 . Preferably, the groove  22  for gate placement is located at each open end  16 , but may be located at only one end  16 , or may be placed at any other point along the length of the casting tray  10 . Preferably, the groove  22  is precision machined or otherwise formed to create a fluid-tight fit with a casting gate  24 . 
   The casting tray  10  can be made to any dimension with preferred dimensions comprising a width of at least four inches and a length of at least nine inches. For longer gels, the length of the casting tray  10  is preferably at least twelve inches, and more preferably, greater than fifteen inches. The depth of the sidewalls  14  is preferably at least one inch. 
     FIG. 2  is a perspective view showing the casting tray  10  with one open end  16  sealed by means of a casting gate  24  and the other sealed by a slidable block  26 . The casting gate  24  is made of acrylic, aluminum, silicone rubber or any other material capable of forming a fluid-tight seal when placed within a receiving groove  22 , and of retaining a substantially flat shape upon repeated use. The slidable block  26  is preferably made of a material with high thermal conductivity, such as aluminum, alumina, brass, or silver, or other suitable material, so that when molten agarose is used as a separation medium, heat is rapidly absorbed allowing the agarose to gel quickly and seal the edge adjacent the slidable block  26 . Also, for some applications, for example when using a low concentration of agarose, chilling the block beforehand in a freezer or refrigerator can enhance gelling. Alternatively, the slidable block  26  may be made of any material or composite of materials that permits the formation of a fluid-tight seal. 
   The slidable block  26  preferably has a knob  28  positioned anywhere along its length to facilitate sliding of the block  26 . Alternatively or in addition, the slidable block  26  may be provided with one or more indents or depressions  30  to accommodate finger placement to facilitate sliding of the block  26 . Likewise, a ridge or ridges on the top surface of the slidable block  26  can be used to facilitate sliding (not shown). The knob  28  or finger placement depressions  30  or ridges are not necessary for sliding the block  26 , and thus, the block  26  may be formed without any feature to facilitate sliding. The knob  28  is preferred when using the block  26  to prepare step gradients as shown in  FIG. 6 . The slidable block  26  is preferably at least one-half inch in thickness. 
   Referring to  FIG. 3 , a perspective view of a conventional comb  20  used to form sample wells is shown. Combs  20  may be made of polycarbonate, acetal, polyetherimid, or other materials that are well known in the art for such applications. The teeth  32  of the comb  20  form slots or wells  36  in a separation gel  34 , as shown in  FIG. 5 , to be filled with a sample preparation, calibration standards or other liquid specimens. The teeth  32  may be varied in thickness, width, and number as desired. 
   A casting tray  10  with a casting gate  24 , a slidable block  26 , and a comb  20  in place within a set of notches  18  on opposing sidewalls  14  of the casting tray  10  is shown in  FIG. 4 . More than one comb  20  may be used, if needed, to form wells  36  within the body of a solidified separation medium  34  to accommodate increased sample numbers. The slidable block  26  is shown in close proximity to the comb to form a relatively short solidified separation medium, but the slidable block  26  may be placed anywhere along the length of the casting tray  10 . 
   In use, preferably, one end  16  of the casting tray  10  is sealed with a casting gate  24  seated within a groove  22  in the casting tray  10 , and the other end  16  is sealed with a slidable block  26 , as shown in  FIGS. 4 and 5 . The length of the separation medium is varied by placement of the slidable block  26  along the length of the casting tray  10 . Before or after a separation medium  34  is poured, one or more combs  20  are placed prior to solidification of the medium. When the medium  34  is set, the combs  20 , casting gate  24 , and slidable block  26  are removed for use in an electrophoresis device (not shown) as is conventional in the art. Alternatively the slidable block  26  can remain in the casting tray  10  and merely moved away from the bottom of the solidified separation medium to allow contact with an electrolyte-containing buffer. For such use, the slidable block  26  may be anodized or “hard-coated” to make the block  26  electrically non-conductive so that the base metal does not corrode electrolytically with repeated use. 
   To form a gradient gel, the slidable block  26  may be initially placed close to the end  16  that is sealed with a casting gate  24 , as shown in  FIG. 5 , at a desired distance from the gate  24 , and a volume of medium containing a first concentration of agarose or other separation component can be added. After the medium solidifies  34 , the slidable block  26  can be moved incrementally as desired along the length of the casting tray  10 , and separation medium of a second concentration can be added. These steps can be repeated as needed with additional concentrations of separation medium to form the desired gradient. 
   Alternatively, a simple “step” gradient is formed more quickly through the use of two or more slidable blocks  26 . Referring to  FIG. 6 , the two open ends are sealed with casting gates  24 , sealing tape, or any other suitable means of preventing leakage, and the slidable blocks  26  are placed with a spaces or gaps  38  of desired length between the two blocks and between the blocks and the casting gates  24 . A different concentration of separation medium is poured into each of the gaps  38 . Referring to  FIG. 7 , when the medium in the gaps  38  solidifies, the blocks  26  are removed, creating voids  40  where the blocks  26  had been. Solutions of separation medium with different concentrations from those previously used and from each other, are then poured into the voids  40 . As an example, the gaps  38  formed by initial placement of two slidable blocks  26  as shown in  FIG. 6 , can be filled with solutions containing 0.5% and 1.5% agarose w/v respectively, then when the blocks  26  are removed as shown in  FIG. 7 , the voids  40  can be filled with solutions containing 1.0% and 2.0%, respectively. The blocks  26  may be independent of one another, or alternatively, two or more blocks may be connected, preferably by a handle attached to the top surface of each (not shown), in a fixed pattern. 
   Other embodiments, modifications, improvements and alternatives may be apparent to those skilled in the art. Such embodiments, modifications, improvements or alternatives are considered to be within the spirit of the invention. 
   Various alternatives and embodiments are contemplated as being within the scope of the following claims particularly pointing out and distinctly claiming the subject matter regarded as the invention.

Technology Category: 7