Chemistry: electrical and wave energy – Apparatus – Electrophoretic or electro-osmotic apparatus
Reexamination Certificate
1997-09-30
2001-05-15
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Apparatus
Electrophoretic or electro-osmotic apparatus
C204S619000
Reexamination Certificate
active
06231741
ABSTRACT:
The present invention relates to a gel cassette and an electrophoresis apparatus to be used therewith.
The separation of different biological materials, such as proteins, RNA and DNA generally takes place by means of electrophoresis. This technique is based on the measurement of the mobility of molecules in an electrical field. The migration speed of a molecule in an electrical field depends on the electrical field strength, the net electric charge on the molecule and the frictional resistance. When a detergent such as sodium dodecyl sulphate (SDS) is added to the gel, the separation is only based on differences in size.
An electrophoretic separation takes place in a gel, for instance an agarose gel or a polyacrylamide gel. Particularly these latter gels are very thin and are manufactured by causing a still liquid polyacrylamide solution to polymerize between two glass plates. The thickness of the gel is determined by the so-called spacers, which are situated between the glass plates.
Because the starting point is a gel solution which in the first instance is liquid, the space between the glass plates must be a leak-free compartment, since otherwise the gel solution would immediately run out again from between the glass plates. A gel cassette consists for this purpose of two glass plates, two side spacers and a plug which closes off the underside of the space. The spacers on the side parts of the gel have a dual function, on the one hand in defining the casting compartment and on the other hand for an electrical insulation of the gel between the glass plates relative to the buffer outside the glass plates.
The underside of the glass plates is closed off before casting the gel. Use can be made herein for instance of an agarose plug or a polyacrylamide plug. The advantage hereof is that the gel cassette consisting of the glass plates and the side spacers can be mounted directly into the electrophoresis apparatus and can also remain there. The great drawback however is that casting of a gel in this manner takes a great deal of time. In addition, the agarose plug has the drawback that the electrophoresis process can be slowed thereby.
It is further also possible to arrange a sealing profile on the underside of the gel cassette, wherein leakage of the liquid gel solution from between the glass plates is prevented by means of clamps. The use of tape for sealing is often also necessary herein. Even then, however, a leak-free cassette is not always obtained. This method of working moreover has the drawback that arrangement and removal of the tape require extra operations. Furthermore, the gel cassette can only be placed in the electrophoresis apparatus after polymerization of the gel, because the sealing profile, the clamps and the optional sealing tape can be removed prior to electrophoresis but only after polymerization of the gel.
The present invention has for its object inter alia to provide a gel cassette and electrophoresis apparatus with which the above mentioned drawbacks can be obviated and with which it is optionally also possible to determine whether the buffer still functions properly.
The invention provides for this purpose a gel cassette, comprising two glass plates with a substantially H- or U-shaped spacer for placing therebetween. When a substantially H- or U-shaped spacer is used, both sides and the underside of the space between the glass plates are sealed by the spacer during casting. The two vertical side parts of the spacer preferably protrude above the glass plate. After polymerization of the gel solution between the glass plates, the vertical parts of the spacer are pushed downward, whereby the spacer will drop and the underside of the gel will be released. Hereby occurs the required electrical contact between the underside of the gel and the surrounding buffer. The side of the gel continues to remain isolated from the buffer by the spacer. Underneath the gel there remains a small space between the glass plates where no gel is present.
Using the gel cassette according to the invention it is possible to create rapidly a leak-free space between glass plates, into which the gel can be cast.
In an alternative embodiment the invention provides a gel cassette which can be used to cast so-called perpendicular gels. These are gels in which the gradient runs not in vertical but in horizontal direction. For this purpose at least one of the parts of the spacer placed vertically in the situation of use consists of two portions mutually connecting in liquid-tight manner to define a passage opening for gel solution. For casting of the gel, the gel cassette is rotated a quarter-turn, whereby the divided part of the spacer is situated at the top. By pushing apart the two parts a passage opening is formed, through which the space defined by the spacer and the glass plates can be filled with gel solution. After polymerization the gel cassette can be placed upright again and electrophoresis can take place.
When a comb with two wide teeth is used, two gels can be run simultaneously. During casting one gradient gel is cast first, whereafter a narrow segment of gel without gradient is arranged in order to separate the two gradient gels from each other. The second gel can then be cast.
The ends of the two portions of the vertical spacer part which connect mutually in liquid-tight manner are preferably toothed but mutually fitting concave and convex or step profiles are also possible. The advantage of a toothing is that the mutually abutting positioning of the two portions then proceeds easily.
To ensure a good sealing at the position of the comb which functions as a side during casting of the gel, the invention provides as extra accessory with the gel cassette a pressure clamp comprising a U-shaped element and a pressure element protruding through one of the legs thereof. In order to fix the pressure clamp a recess can be incorporated in the U-shaped element for passage of the hose pillar of the buffer container. The pressure element is for instance a screw which can be provided with a plate on the end remote from the head.
The combination of the divisible spacer and the pressure clamp forms an adapter set for casting perpendicular gels. The adapter set can be used with any desired cassette.
Because a gel shrinks slightly after polymerization it will no longer be connected to the side parts of the spacer. No samples can therefore be arranged on the edge of the gel because due to the space between the gel and the spacer variations may occur in the gradient and the electrical field and therefore in the band pattern. In order to prevent this the parts of the spacer substantially vertical in the situation of use are wider on their end remote from the part of the gel substantially horizontal in the situation of use. In practice this means that in the case of a spacer which is pressed downward the upper sides of the vertical spacer parts are wider, while in the case of a spacer which is pulled upward, such as a spacer with comb, it is on the contrary the lower ends of the vertical parts which are wider. The outward facing edge of these vertical parts preferably runs straight, while the inward facing edge can taper toward the outer end in a convex, concave or linear manner.
During casting of the gel air bubbles can develop at the bottom, which are difficult to remove and can cause malfunction of the gel. To prevent this the spacer is preferably provided with a shoulder at the position of the connection of the horizontal part onto the vertical part. By tilting the gel cassette the air bubble will rise along the horizontal part of the spacer in the direction of the vertical part of the shoulder. Having arrived there it will move along that vertical shoulder part and finally along the horizontal shoulder part to ultimately come to a stop against the vertical spacer part. When the gel cassette is placed upright again the air bubble will no longer be able to return to the bottom of the gel, since that would be a downward movement which will never be made by an air bubble. The air bubble can pos
De Vos Gerrit J.
Tuurenhout Matthijs W. F.
Venema Paul
Banner & Witcoff , Ltd.
de Vos Gerrit J.
Noguerola Alex
Warden Jill
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