text:
A method of isolating a biological material by
a) providing a biological material which is bound to a porous matrix (C11), and
b) compressing the matrix under conditions where the biological material is released from the surface of the matrix into an elution liquid.
brings about the advantage that the emission of aerosols into the environment is greatly reduced. It is also possible to obtain highly concentrated and minute amounts of solution. The method is also easy to automate.
1. A method for isolating a biological material, comprising:
providing a hollow structural form having a first opening and a second opening and comprising an elution liquid-insoluble solid compressible porous matrix;
binding a biological material to the compressible porous matrix;
thereafter introducing the hollow structural form into an elution liquid-containing elution vessel through an opening therein, wherein the outer wall surface of the hollow structural form urges against the inner wall surface of the elution vessel, and wherein the first opening and the compressible porous matrix are configured to allow the passing of the elution liquid from the exterior of the hollow structural form through the first opening and into the compressible porous matrix;
thereafter inserting a piston, having a contact surface, into the hollow structural form through the second opening therein, and compressing the compressible porous matrix using the contact surface to force any elution liquid and biological material bound by the compressible porous matrix above the contact surface of the piston; and
locking the contact surface in place using locking means, provided on the piston, for maintaining the compression of the compressible porous matrix by the contact surface.
2. The method of claim 1, wherein the biological material is a nucleic acid.
3. The method of claim 1, wherein the compressible porous matrix comprises glass fleece.
4. The method of claim 1, wherein the piston defines a hollow interior and has a first opening adjacent the contact surface, wherein the hollow interior receives the elution liquid and biological material forced from the compressible porous matrix through the inlet opening.
5. The method of claim 4, wherein the piston has a second opening configured to allow the removal of the biological material from the hollow interior, and the method further comprises, after the locking step, removing the biological material through the second opening without unlocking the locking means.
Subject matter of the invention is a method of isolating a biological material by binding said biological material to a solid phase, and releasing said material in a special procedure and a system suitable for isolating said biological material.
Recently, biological materials have gained increasing interest in many fields. This is facilitated by the fact that it has become possible over the last few decades to separate biological materials from other materials. Biological materials are generally available in a complex mixture together with other materials. Moreover, most biological materials are also present in very minute amounts compared to other components of a biological individual. Changes of biological materials with respect to its normal condition serve to diagnose the condition of the biological individual. Methods of analyzing biological materials are, hence, of particular interest in the field of molecular biology and health care. Depending on the required analysis, a more or less specific method of isolation is selected. There exists a multitude of isolation methods for biological materials which depend on the type of biological material to be isolated and its subsequent use. In the method used in the analysis of antigens and antibodies, the biological material (e.g. an antigen, antibody or nucleic acid) is bound to the non-porous inner wall of a cuvette made of glass or polystyrene. In this case, the binding of the biological material is so specific that the biological material to be detected is immobilized on the surface. This method does not propose to again release the biological material; this would even be adverse to the subsequent quantitative determination.
A second type of method uses swellable, porous materials to separate biological materials, e.g. according to their molecular weight. In this method, there is no binding between a biological material and a solid phase. The separation is essentially accomplished by using the different penetration properties of biological materials based on their different sizes.
A third kind of isolation method is based on the varying degree of binding of different biological materials to porous materials which exhibit a particular affinity to the biological material when specificity-enhancing groups are attached. This method makes use of column-like accumulations of particle-type affinity materials. A liquid which contains the biological material is made to pass through this accumulation of material. The biological material to be isolated is then bound to the surface of the porous particles on the groups with the affinity for the biological material; all other components emerge from the column together with the remaining liquid. In a subsequent step, the biological material is released from the porous material by allowing an elution to flow through the column in exactly the same direction which then releases the binding to the porous material. The biological material is then contained in the elution liquid.
In presently known methods, the elution liquid was passed through the porous matrix either by applying pressure in flow direction of the liquid, or even by centrifuging the column to spin the elution liquid out of the porous matrix. This method, however, requires the use of vacuum pumps or centrifuges, hence, instruments that are often used in medical routine diagnostics for this particular purpose. Moreover, the use of these instruments, especially centrifuges, is time-intensive and does not allow the continuous processing of samples.
It is an object of the present invention to provide a new method of isolation for biological material which brings about improvements with respect to those known in prior art.
Subject matter of the invention is a method of isolating a biological material by providing a biological material bound to a compressible porous matrix, and compressing the matrix under conditions where the biological material is released from the surface of the matrix into an elution liquid. Another subject matter of the invention is a system for isolating the biological material.
An isolation method as understood in the invention is a method where one or several components of the mixture are separated from the remaining components of this mixture. This covers in particular those methods where the one or several components to be separated are bound to a solid phase, where the remaining liquid is removed and the one or several bound components are subsequently released into another liquid.
Biological material is understood to be organic compounds that are in a relation to beings such as animals, humans, viruses, bacteria, or plants. They include, for example, the components found in such beings. Particularly preferred components are those that are present either in a dissolved form or can be dissolved in liquid, but can also be bound to a solid matrix. They include low-molecular substances, on the one hand, (with a molecular weight of less than 2000 D), such as vitamins, but also therapeutically active substances and hormones; further, they also include high-molecular substances (with a molecular weight of more than 2,000 D), such as biopolymers that consist of monomer units. They include, for example, proteins and nucleic acids. The method in accordance with the invention is particularly suitable for isolating nucleic acids. When dealing with proteins, the immunologically active substances, such as antigens and antibodies are particularly preferred.
Biological materials which can be isolated in a preferred manner are recovered from a liquid of the being in question and bound to a matrix. Depending on the part of the being from which the biological material is to be isolated, the being may be subject to a pretreatment. Such a pretreatment serves to release the biological material from the being. Such a pretreatment is necessary, for example, when the being is a bacterium or a group of bacteria. In this case, it is preferred to destroy the cells to allow the components of the being to be released into the liquid. Subsequently, the solid substances that may have formed can be separated. If the biological material is present in a liquid in an already accessible form, e.g. in a body fluid, cells or other substances that may interfere with the isolation of the biological material are often separated. This can be accomplished by means of filtration or by using affinity materials. In any case, the result of a pretreatment is always a sample liquid which contains the biological material in a form where it can be bound to a matrix.
In one step of the method of the invention, the biological material is provided in a form where it is bound to a compressible porous matrix. The matrix as understood in the invention is a non-soluble material present in the liquid containing the biological material. The chemical composition of the matrix is determined by the fact that the matrix must be compressible and porous, for which reason organic or inorganic polymers are used. Organic polymers include, for example, plastics such as polystyrene, but also cellulose, e.g. paper. Inorganic polymers are in particular substances which contain a certain percentage of glass. The matrix, can, however, also consist of a metal.
The compressible porous matrix in accordance with the invention is a spatially extending structure having a non-soluble part made of the above-mentioned material and a part which can be filled with liquid. This fillable part is in the following referred to as the inner volume. The inner volume extends between the non-soluble part thus forming a system of coherent porous and/or empty spaces. This system could preferably also be referred to as an open pore system.
The non-soluble part in the system also forms a spatially extending structure, which, in a preferred manner, has a coherent structure. It is possible, but not preferred, that the matrix be a multi-component system, e.g. a system where the particle-type component is sealed between two particle-type components in direction toward the interior (C14) and in direction of a liquid-permeable component, e.g. a fleece, at the lower opening in the structural form. The particle-type component can then assume the function of a slurry between the two components; in order to obtain the liquid, the two components are added to one another which then leads to a compression of the slurry and consequently reduces the liquid volume contained therein.
Examples for such a compressible porous matrix are sponge-type structures and structures consisting of fibers such as fleeces. In a preferred manner, the matrix is capable of absorbing liquid, it is also possible to apply sample liquid to its surface.
A compressible matrix as understood in the invention is one where the inner volume can be reduced by 50% or more, preferably 70% or more, or particularly preferred 90% or more, by pressing together the spatially extended structure of non-soluble part and inner volume. For practical reasons, it is unlikely that a complete elimination of the inner volume can be achieved; a certain percentage of inner volume and, hence, liquid will therefore remain in the matrix.
The percentage of volume of non-soluble material to inner volume preferably ranges between 10:1 and 1:100, particularly preferred between 1:1 and 1:50.
The compressibility of the porous matrix must not necessarily be reversible; i.e. when releasing the pressure exerted on the matrix, the latter must not necessarily resume its original shape. The extension of the matrix depends inter alia upon the amount of biological material to be bound. The outer form can be freely selected. It depends inter alia upon the type of immobilization of biological material and the subsequent release. A plate-like form of the matrix is preferred.
The non-soluble material of the matrix can already be selected such that the biological material exhibits a high affinity to this material, thus facilitating binding. Such a case is given for the immobilization of nucleic acids when a glass fiber fleece is used as the non-soluble material. It is known that all nucleic acids exhibit a certain affinity to glass surfaces. When the material itself does not exhibit any affinity to the biological material to be isolated, the surface of this material can be modified so as to allow binding. In the case that nucleic acids are to be bound, it is possible to bind so-called capture probes to the surface, i.e. nucleic acids which have a sequence that is complementary to the nucleic acid to be isolated. When immunologically reactive compounds are to be isolated, i.e. antigens or antibodies, it is possible to immbolize the corresponding immune partners, i.e. antigens or antigens. The binding of capture probes and/or immunoreactive compounds can be accomplished with the aid of methods that are also known for non-porous materials.
The biological material bound to the compressible porous matrix can be accomplished in that the matrix is brought into contact with the solution of the biological material and incubated for a sufficient period of time. Since the amount of solution in which the biological material is present usually exceeds the inner volume of the matrix, it is preferred to allow the solution to pass through the matrix, e.g. by drawing off or centrifugation. In a particularly preferred manner, the solution is pressed through the matrix in the vessel without applying low pressure or centrifugation. In a preferred manner, the liquid present in the interior of the matrix containing the components of the solution to be bound is subsequently removed from the matrix, e.g. also by drawing off, centrifuging or pressing. If desired, the matrix which now contains the biological material in a bound form can be washed and contamination that may adhere to it can be removed.
The core of the invention is the manner in which the biological material is released from the matrix. Owing to the fact that the matrix is compressible and porous, a solution of the isolated biological material can be obtained by compressing the matrix under conditions where the biological material is released from the matrix surface into an elution liquid. Compression means compressing the spatially extended structure together and thus pressing the liquid out of the inner volume of said structure. This increases the percentage of non-soluble material in the total structure while the percentage of the remaining inner volume is reduced. As proposed by the invention, the matrix is reduced by more than 40% and particularly preferred by more than 60%. The extent of the compression depends among other things also upon the desired amount of liquid which contains the biological material. The liquid emerging from the matrix can be obtained in any desired manner and be further processed.
An elution liquid as understood in the invention is a liquid where the biological material to be isolated can be dissolved. It has such a chemical composition that the balance between non-soluble material and solution is shifted to the side of the solution. If nucleic acids are then to be isolated, the solution used as elution solution is one with a lower salt concentration than the solution used to bind the nucleic acids to the non-soluble material.
The method of the invention is preferably carried out in a system. To accomplish this, the compressible porous matrix, together with the biological material bound thereto, is introduced into an elution vessel. The compressible porous matrix is preferably part of a vessel whose inner form matches the interior of the elution vessel. In order to release the biological material, elution liquid is added to the matrix and subsequently compressed with the aid of a piston; the possible outlet for the liquid is provided either in the piston or in the vessel containing the matrix or in the elution vessel or between these components.
The materials used to design the aforementioned vessels are preferably those that can be manufactured in an injection-molding procedure, e.g. plastics such as polystyrene, particularly preferred polypropylene, and optionally with suitable additives.
FIG. 1 is a structural form C configured as a vessel containing a compressible porous matrix in its lower portion (longitudinal section).
FIG. 2 is a system of the invention where the matrix is compressed (longitudinal section).
FIG. 3 is a piston in accordance with the invention to compress the matrix and receive elution liquid (longitudinal section).
FIG. 4 shows an elution vessel D which is advantageously suitable for pressing a compressible porous matrix (longitudinal section).
FIG. 5 is a diagrammatic representation of the operating steps and the structures involved in the method for isolating a biological material in accordance with the invention.
The following description further illustrates the objects listed in the figures in greater detail:
FIG. 1 is a longitudinal section of a structural form (C) which essentially has the form of a hollow cylinder with an upper and a lower opening. In the lower portion, the compressible porous matrix (C11) is fixed in position by tapering the cross section of the tube. Towards the bottom, the matrix is held by an edge (C19) and toward the top by means of a circumferential break-away bar (C18). By providing a desired breaking point along the inner wall (C16) of the structural form, this bar can be broken off and shifted in direction toward matrix (C11). The structural form (C) also has an inner contour (C12) and an interior which is configured as a hollow body (C14). Moreover, this structural form is provided with means (C17) to fix a piston (E) in the structural form. This means is preferably configured such that a piston is fixed in the position in which it compresses the matrix (C11). Moreover, the structural form (C) is preferably equipped with means (C15) to fix a lid on the upper opening. Further, it is also possible to provide means (C13) to fix the structural form in the elution vessel (D).
FIG. 2 is a particularly preferred system in accordance with the invention. It comprises the following components: elution vessel (D), structural form (C), piston (E), and lid (B). Lid (B) is configured such that it covers all the vessels. FIG. 2 shows the system in a form where the elution liquid that is originally contained in the matrix is pressed out of said matrix into the interior C12 of the piston.
FIG. 3 is a particularly preferred piston (E) to compress the porous matrix. It is characterized by a sealing ring (E15) in the lower portion which seals the space between the structural form (C) and elution vessel (D) thus preventing that any solution enters the so-generated capillary gap. In its upper area, piston (E) also has a snap-in ring (E16) which irreversibly fixes the piston in the structural form when the piston is introduced into the snap-in notch (C17) of the structural form (C). The so fixed piston (D) exerts sufficient pressure on the matrix to press liquid out of said matrix.
The pressure exerted onto the matrix can be adjusted in a certain range via the length of the snap-in connection of piston E and the position of the snap-in notch in the structural form (C). The lower geometry of piston (E) shows a pressing piston whose form-fitting geometry of the contact surface (E10) is suitable to ensure proper compression of the matrix. The form of the piston is configured such that the dead volume is minimized. Recesses (E17) act as a receptacle for broken off bars (C18).
In order to obtain and collect the solution pressed out of the matrix, the lower portion of the piston is provided with a transversal cylindrical bore; said bore has a relatively small diameter and a height that is suitable to receive the nucleic acid-containing solution that was pressed out, but also to remove liquid by means of a pipette. FIG. 3 shows a configuration where bore (E 18) is relatively narrow with respect to the possible internal volume of the piston. The one side of bore (E18) ends into opening (E13) in the contact surface and the other side into the interior of piston (E12).
The upper area of the piston is also a transversal bore which serves as a receptacle for the solution of biological material obtained; as compared to the lower part, it has a relatively large diameter and a suitable height. Diameter and height are selected such that they are suitable for introduction of the pipette into the interior (E12) and to remove liquid that has entered the interior via bore (E18).
FIG. 4 is a longitudinal section of a preferred elution vessel (D). It has an inlet opening (D10) for the introduction of liquids and/or other functional units into the elution vessel. Moreover, it has an inner edge (D11) to attach a lid (B). A snap-in notch (D12) is also provided in the upper part of the elution vessel; said notch serves to position the structural form via means (C13). Another preferred feature are means for attaching the elution vessel (D13) in an apertured plate of a suitable diameter. A particularly preferred feature is also the contact surface (D14) in the lower part of the elution vessel. By matching this surface to the lower part of the structural form, it is possible to reduce the dead volume inside the vessel. This leads to a particularly effective recovery of biological material.
In a particular embodiment of the method of the invention for processing nucleic acid-containing sample solution, the following operating steps are carried out (see FIG. 5). In a first step (I) a cell-containing sample liquid is incubated in a sample vessel (A) with a material to which the cells are bound in order to obtain the nucleic acids. To accomplish this, said material can either exhibit specific binding properties for the surface of the cells, e.g. by immobilizing antibodies to surface antigens or an absorber material (A16, not shown); it is, however, also possible to provide a material with filter properties (A15, not shown) which retains the cells when liquid passes through the material, e.g. when removed from the sample vessel. Conditions for immobilizing cells on surfaces are known to the expert, e.g. from Methods in Enzymology, vol. 171, Biomembranes/Part R Transport Theory: Cell and Model Membranes, edited by Sidney Fleischer, Becca Fleischer, Department of Molecular Biology, Vanderbilt University, Nashville, Tenn.
During incubation, a lid (B) preferably closes the sample vessel to ensure active and passive protection from contamination.
In another step, the liquid is removed from the sample vessel while cells whose nucleic acids are to be isolated remain in the vessel where they are bound to the material. If the cell-binding material is a particle-type material, the cells can be retained in that the material is magnetic and a magnetic field is applied to the sample vessel from the outside; said field has to be strong enough to retain the particle-type material in the sample vessel when the liquid is removed. The liquid can be removed in different ways. It is, for example, possible to remove the liquid through an outlet opening (A11) which is spatially separated from the inlet opening (A10). If said outlet opening is located in a lower part of the sample vessel and below the retained cells, the liquid can be drawn off, e.g. by applying a minor vacuum. To accomplish this, a valve may be provided at the outlet opening to generate such a low pressure.
In order to remove other potentially interfering sample components from the cells, it is possible to provide one or several washing steps. To achieve this, washing liquid is filled into the sample vessel; said washing liquid dissolves possible contamination which, however, does not essentially affect the binding of the cells to the surface of the cell-binding material. Such washing solutions are known to the expert, e.g. from cell-separation protocols or corresponding cleaning kit protocols for nucleic acids. They basically depend on how the cells bind to the material.
After the last washing solution has been removed from the sample vessel (A), the purified, enriched cells are brought into contact with a suitable lysis liquid to release the nucleic acids from the cells. The reagents of this lysis solution largely depend on the type of immobilized cells. If the cells are bacteria, the lysis solution preferably contains proteinase K to digest the cell walls. Optionally, the lysis can be supported by heating or cooling and agitating the reaction mixture. If magnetic particles are used as cell-binding material, the mixing can also be accomplished with the aid of a magnet. Moreover, it is possible to mix the solution by shaking the sample vessel. Once digestion is completed, the nucleic acids to be isolated are freely accessible in the solution.
Even during lysis, it is preferred that the reaction vessel be closed by a lid in order to avoid contamination from the environment. After completion of the lysis, the lid is removed, preferably with the aid of a corresponding mechanical device. Subsequently, a structural form (C), whose outer contour (C12) matches the inner contour (A17) of the sample vessel, is introduced into the sample vessel which contains a mixture of digestion products of the cells and the nucleic acids. This structural form is hollow and sealed toward the sample vessel and toward the reaction mixture by means of a filter. The introduction of the structural form (C) is preferably accomplished with the aid of a component (B11) of lid (B) which also contains a component (B10) suitable to close the sample vessel. In this case, the structural form is taken up with the aid of a lid (II) and introduced in the sample vessel while the latter is closed. During this procedure, the reaction mixture can enter the hollow space (C14) of the structural form across filter (C11) (IV). By providing a filter, it is possible to prevent large particles from entering into the hollow space; if the filter already has nucleic acid binding properties, the nucleic acid can already be bound to the filter while the reaction mixture is passing through. In this case, it is expedient to select a glass fiber containing filter material.
In the next step, the remaining lysis reaction mixture is removed from the device consisting of A and C, e.g. by drawing off solution from the sample vessel through an outlet opening (A11) located in the lower portion of the vessel. The solution that has entered the hollow body (C14) of the structural form is, hence, also removed so that the filter more or less no longer contains any liquid. Subsequently, the so far used lid (B) is removed while the structural form (C) still remains in the sample vessel (where it is snapped into position) (V).
Simultaneously or subsequently, an elution vessel (D) is prepared to receive the structural form (C). A lid that can be provided on this vessel, if necessary, is removed (VI). Preferably, an elution solution is provided, e.g. by pipetting, in the elution vessel prior to transferring the structural form (C) into the elution vessel (D). The composition of the elution solution depends on how the nucleic acid is bound to the material in filter (C). It contains reagents which cause the immobilized nucleic acids to elute from the material, i.e. to be released therefrom. Lid (B) initially used to close the elution vessel, is now placed onto the sample vessel (A) with the structural form (C) (VII).
In order to take the structural form (C) out of sample vessel (A), the lid is first removed. The combination of lid and structural form is subsequently introduced into the elution vessel. In a preferred manner, the structural form (C) contains a means (C13K) to fix the structural form in position in the elution vessel (D). Owing to said means, the structural form (C) or the vessel (D) have to be destroyed in order to remove said structural form, or a force has to be applied which exceeds the force necessary to remove the lid (B) from structural form (C). The invention does not propose to remove the structural form from the elution vessel.
While the structural form (C) enters the elution vessel, already provided elution solution enters the matrix (C11) to release the immobilized nucleic acid from the solid matrix. Depending on the amount of prepared elution solution, either only the filter is impregnated with the elution solution or the elution solution enters the hollow body (C14) together with a released nucleic acid. For complete elution of the nucleic acids, the inner contour of the elution vessel should be configured to urge as tightly as possible against the outer contour of the structural form.
In a subsequent step, lid (B) is removed from the combination of structural form (C) and elution vessel (D) (X). Said lid (B) is used to take up a piston (E) (XI) and introduce said piston (E) into the hollow space of the structural form (C) (XII). Said lid engages piston (E) in the inside. The piston is pressed against the filter (C11) such that liquid which is present in the filter passes through an opening located in the contact surface into the interior of the piston. This procedure is particularly effective when the outer contour of the contact surface matches the inner contour of the structural form (C) in at least the area where said pressing is accomplished. Piston (E) can preferably be fixed in this position, e.g. by allowing it to snap into position. Since the so formed device is relatively well sealed by means of the lid, the nucleic acid containing solution can be stored in the device.
In order to remove the desired amount of nucleic acid solution, the lid can be removed (XIII) and the desired amount can be taken out via an opening in the interior of the piston, e.g. by means of pipetting (XIV). Subsequently, the lid can be placed back into position.
The method of the invention preferably comprises the following steps:
a) binding the biological material to a compressible matrix (C11)
b) adding an elution liquid into an elution vessel (D)
c) introducing the compressible porous matrix (C11), particularly preferred into a structural form (C)
d) introducing a piston (D) into the structural form (C) and the elution vessel (D); said piston has a contact surface suitable to compress the matrix; the piston has an interior (E12) to receive the liquid pressed out of the matrix
e) allowing the piston to engage the structural form so that the matrix remains compressed.
The sequence of steps b and c may be exchanged in this procedure, if desired. The elution liquid containing with biological material which has entered the interior can subsequently be stored in the system (preferably with the lid being closed); or it can be removed with a pipette and processed in any desired way.
The method of the invention is particularly suitable for generating solutions of biological materials with relatively high concentrations. The invention allows the generation of nucleic acids from a matrix while the generation of aerosols is reduced. Moreover, the operating steps as proposed in the invention greatly facilitate automation of the process and method of isolating biological materials.
Another subject matter of the invention is a system for isolating a biological material which comprises the following components:
an elution vessel (D)
a compressible porous matrix, and
a piston (E) to compress a matrix
Additional preferred features are described in the method of the invention.
The following examples describe the invention in greater detail.
A preferred embodiment of the piston is a hollow cylindrical plastic tube (E) having different diameters in its interior (D [bottom]=1 mm; D [top]=5 mm).
The piston can be characterized by the following dimensions:
The lower part of the piston is provided with a central bore (E 13); the top is receptacle (E 12) which is open toward the top.
At its top, the outer surface (E) has snap-in lips (E 16) to mount and position structural form (C). The circumferential snap-in lip is located 3,5 mm below the upper edge; it has a depth of 0.3 mm and a height of 0.25 mm. In its lower part, the piston is provided with sealing lips (E 15) which seal the piston with respect to the structural form. Said lips are elevated by 0.28 mm and have a width of 0.1 mm.
The upper opening of the piston has an inner geometry to link a functional lid (B) to an elution vessel (D). Piston (E) is configured such that it can be introduced into the structural form C to close together with lid B the entire system.
Biological materials/chemicals/devices
Sample vessel (A)
Lid of sample vessel (B)
Structural form (C) having a porous matrix made of a glass fleece (C11)
Elution vessel (D)
Lid of elution vessel (E)
Pressing piston of structural form (F)
Preparing the sample solutions
How an example is carried out
Sample vessel (A) is filled with 200 μl of sample, 25 μl of proteinase K solution and 200 μl of binding buffer. Sample vessel (A) is closed with lid (B) of the sample vessel. The liquids are mixed in the closed vessel. Subsequently, the mixture is incubated at 70° C. for 10 min. (digestion of cells), followed by a cooling phase where it is cooled down to 20° C. in 3 minutes. Sample vessel (A) is opened, and 210 μl of ethanol are added. Sample vessel (A) is closed, and the solution is mixed.
Sample vessel (A) is opened. The lid (B) of the sample vessel is used to get the structural form (C) with a porous matrix out of its support. The structural form with the glass fleece matrix is introduced into the sample vessel which is filled with liquid through the inlet opening. While being introduced, the liquid present in the sample vessel (A) passes through the glass fleece from the bottom. The freely moving nucleic acid now binds to the glass fleece matrix. In the next step, the liquid present in the structural form (C) is drawn off towards the bottom. The liquid again passes through the glass fleece and nucleic acid which is not yet bound is now immobilized on the matrix.
The glass fleece matrix is washed twice with 500 μl of washing buffer. The buffer solution above the matrix is drawn off through the glass fleece. Subsequently, the matrix is dried with a purified nucleic acid at 50° C. for 3 minutes. Lid (B) of the sample vessel is discarded.
Using lid (E) of the elution vessel, the structural form (C) containing the nucleic acid is transferred from the sample vessel (D) into a prepared elution buffer with 200 μl elution buffer in it. The elution buffer releases the nucleic acid from the glass fleece matrix. The nucleic acid is partially located above the matrix.
By introducing a pressing piston (F) with the aid of the lid (E) of the elution vessel, the matrix in the structural form is compressed in order to minimize the dead volume of the nucleic acid solution.
Nucleic acid yields
The tests were carried out with two different nucleic acid sample concentrations. The following results could be determined:
Patent Citations
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The Fukushima Cleanup Wasted Nearly £337 Million on Bad Technology
That's a third of the total cleanup budget, all wasted on useless tech.
That's a third of the total cleanup budget, all wasted on useless tech.
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More mystery leaks raise even more questions about the incredible complexity of cleaning up a broken nuclear plant.
Fukushima is Japan's radiation nightmare that just won't go away.
The panic over the leakage was instantaneous in the US—but a new study shows whether it was valid.
The host of Veritasium, a YouTube channel about science, recently visited the most radioactive places on Earth for a TV show about how Uranium and radioactivity affected the modern world. And he lived to tell the tale.
Made fresh each day by flown-in mother.
Nuclear cores are obviously shielded from radiation, meaning conventional X-Rays can't pass through to show use what's inside. Fortunately Muon particles can penetrate the shielding.
The Tokyo Electric Power Co. (TEPCO) has admitted that an initial, smaller, experimental inner ice wall is proving difficult to construct.

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Available real estate listings that are similar to 306 Hersey Cres
306 2nd AVENUE W, Lafleche, SK: $160,000.00
306 Main STREET, Gerald, SK: $119,500.00
306 Pine DRIVE, Tobin Lake, SK: $268,000.00
306 31510 GILL AV, Mission, BC: $395,730.00
306 Husky AVENUE, Air Ronge, SK: $360,000.00
306 Douglas AVENUE, Manitou Beach, SK: $75,000.00
306 ELIZA Street, MEAFORD, ON: $94,900.00
306 - 108 SPRUCE AVE, Tumbler Ridge, BC: $108,000.00
General Information on 306 Hersey Cres and surrounding area
This real estate property is located at 306 Hersey Cres in Bolton Towncentre (Bolton). Other areas in the vicinity are: Bolton Rural, Bolton Station, Tormore.
306 Hersey Cres is mostly surrounded by single-detached properties with Scottish population who owned a home the most dominant in the area.
Bolton has 101 homes and condos for sale. Of those, 52 real estate listings are currently available in Bolton Towncentre. The average house price in Bolton right now is $913,000.


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Cabinet depth - upper
I know standard depth for uppers is 12" I also know some people have suggested to get deeper cabinets if money allows.
My question is, is that deeper cabinet depth always 15", or does it vary? Do some people go with 14" or 16" or ???
I currently have 12" in the plan and then the corner easy reach cabinet is 15" to give some depth to the cabinetry.
I'm wondering if I upgraded to deeper cabinets, would I go with 15" and then 18", or 14" and 17"?
Is there any downside of deeper uppers (other than cost of course)? I really only have 5 uppers that I'd do this with. My other wall of cabinets I would leave as 12/15 since it's more like a hutch area (glass cabinets/shelves in the middle cabinet).
Are you saying that you want to stagger the depths on the same wall? If so, then I'd say it depends on what the overall flow is re. how deep you go on the uppers.
We did 13" uppers over 26.5" bases on our range wall, and 16" uppers over 25.5 bases on our breakfast bar wall. Neither are any problem to work at, and the deeper cab stores lots and lots of dishes and glasses.
If you are staggering depths of uppers alone, none of which come down to the cabs, I'd be tempted to do 14/17 or even 13/16. If some of the uppers come down to the base, or if your base cabs are particularly deep, then 15/18 would probably be best. Hard to say for sure without seeing your plan though.
We are planning on 14" uppers - just enough extra depth to accommodate the stuff that has driven me crazy over the years when I put it into an uppper cabinet because when it was put in, it was just slightly too shallow to fix that additional row.
I don't expect that I will find them too deep - I don't lean forward that much when I'm prepping something on the counter anyway, although I am on the shorter side (5'3") so it may not be as necessary for me to lean forward as some taller people might.
Thanks to all for taking the time to reply!
taggie - yes, my cooktop wall runs into the sink wall (they are perpendicular) -- I was going to do that corner cabinet at 15", so it juts out a few inches from the adjacent 12" wall cabinets on the cooktop wall and sink wall --- this would be for looks only - to add some depth to the cabinetry.
elofgren -- well I taped a ziploc box to the cabinet!! Not an issue at all! Thanks for the suggestion!
Now, I'm thinking maybe I'll do all 15" cabinets, including that corner (as currently planned) and then maybe do frosted glass in the 30" cabinets that are on each side of the hood (stainless, not cabinetry). I'm just looking for some good design ideas to make it look good. I'll have a big 60" counter height bumped out window along the sink wall, so I'm thinking that's enough interest along that wall (i.e. no need for glass doors).
I'm flying by the seat of my pants, if it's not obvious.... my cabinetmaker has got to be thinking I'm crazy! :)
Ditto GWLolo. Love the 30" countertops and deep uppers. Recently, DG re-engineered an upper cabinet for vertical storage for my 3 large serving bowls.
These bowls (not the 18" cabinet):
Now they fit very nicely in here:
I ordered a NXR range today and was looking through the installation manual. They specify a maximum of 13" deep upper cabinets. It's not real clear if they're talking about above or next to the range. Anyway, you may need to consider other things (range hoods, built-in microwaves) and also clearance requirements for your particular stove.


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She would join Javier Bardem in pic
Jessica Chastain is in negotiations to co-star with Javier Bardem in "A Most Violent Year."
J.C. Chandor is directing the pic, with Neal Dodson and Anna Gerb producing.
Plot details are being kept under wraps. Chandor also penned the script.
FilmNation is handling global sales.
Chastain, who is repped by CAA and Mosaic, is currently filming Christopher Nolan's "Interstellar."
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150 Years Ago Today…
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- Buck Lawler on 24 March 1865: "No matter if our country goes down tomorrow Lees name will stand first upon the pinnacle of fame, as the greatest of commanders living or dead."
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Tag Archives: winter quarters
7 January 1864: "I wish our Ladies Solidiers Aid Society in Hillsboro would make about fifty prs of gloves for our Company as very few of the men have gloves and they would be very acceptable such weather as this especially on picket."
Item description: Letter, dated 7 January 1864, from James A. Graham to his mother. Item citation: From the James A. Graham Papers #00283, Southern Historical Collection,The Wilson Library, University of North Carolina at Chapel Hill. Item transcription: Camp 27th No. Ca. Inf'y near … Continue reading
Posted in Southern Historical Collection Tagged 27th North Carolina Infantry Regiment, clothes, Hillsborough (N.C.), James A. Graham, ladies' relief societies, substitutes, weather, winter, winter quarters Comments Off


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Carrie Patterson
Carrie has become one of the most recognized and published wedding photographers in the Rocky Mountain West. It is her authentic style and relaxedview profile
Carrie has become one of the most recognized and published wedding photographers in the Rocky Mountain West. It is her authentic style and relaxedview profile
I'm Kristen. Photography has always been a part of my life. My father was an avid amateur photographer, so we always had a darkroom growing up. Some ofview profile
When it came time to name my business I thought about using my name - Emily. But then I thought about the people that know me. The ones who get me. Theyview profile
We are a perfect match if you want to have your cake and eat it too! In essence, you mostly just want to have fun on your wedding day and magically getview profile
Lifestyle photographer Meghan Kay Sadler uses her backgrounds in design and painting to document life with honesty and simplicity. Based in California,view profile
Meg Smith has been described as one of the most distinguished 'light hunters' in the world. Frequently featured in Martha Stewart Weddings, with overview profile
elizabeth is an artist & a mother.....who loves to bring a little more sweetness & beauty into the world.... her innate sense of style isview profile
Spontaneity. Considerations of composition, light and color. Often, the elements of a beautiful, compelling photograph align in the blink of an eye.view profile
Fine art wedding photographer Odalys Mendez has been creating meaningful, compelling imagery for over ten years. Her fresh, simple approach to weddingsview profile
A wedding to me is part photojournalism part, life-style part, directorial. So is my approach to photography. I truly enjoy all the excitement andview profile
My name is Cassidy, a fine art wedding photographer based in Nashville, Tennessee and available for travel worldwide.I'm inspired by beautiful light,view profile
Love is in the air! Please visit www.karenhill.com and fall in love! We love what we do and we love photography. At Karen Hill Photography, ourview profile
Over the last ten years, Gia's work has taken her to weddings traditional and non-traditional, religious and civil, rural and urban, high-end andview profile
At Nancy Ray Photography, we believe in living for what matters most. We believe in creating a legacy with how we live our lives and in the way we captureview profile
Named one of "Martha Stewart Weddings's Top Photographers," the Nichols have been featured in Martha Stewart Weddings, The Knot, Weddings Unveiled, Styleview profile
Karen Wise's modern and sophisticated style is a favorite amongst the elegant New York bride. She specializes in portraiture and draws from her experienceview profile
"I absolutely love weddings! To be able to capture two people at one of the happiest times in their life, couples who have found one another andview profile
Cappy Hotchkiss Photography's vision is classic, modern, emotional and fresh. She achieves dynamic color and striking black and white using both filmview profile
Annie McElwain is a Los Angeles based photographer (though gladly willing to travel). Her photography captures authenticity and emotion, using mostlyview profile
Trent and Dara, the husband and wife behind Trent Bailey Photography, create beautifully authentic and artful images of life's most meaningful andview profile
My path to photography probably started before I was even born, my grandparents met when my grandfather was dropping off film at the local drugstore'sview profile
I tell stories with my cameras. Some of the most important stories I get to tell are that of two people in love and at the heart of these stories are theview profile
After the cake has been eaten, the DJ stops playing tunes, and the beautiful dress comes off, what's left are your memories. Photography plays such anview profile
Britt Chudleigh is an award winning photographer whose formal education as an artist greatly influences her approach to documenting authentic stories andview profile
From Polynesia to Provence, Catherine travels the globe to capture people in love. Photographing on film, her timeless photographs are filled withview profile
Being chosen to tell the story of one of the most real, honest, and loving moments of someone's life is such an honor. Concentrating on natural, intimateview profile
There's no bigger day and, as a photographer, there is no bigger honor. Our studio's love of documenting on film coupled with our passion forview profile
Working together as a fine art husband & wife photography team is an absolute blessing. And, it's one that we don't take for granted for a singleview profile
I am an artist who is inspired by love and life. I absolutely love weddings. It is a great privilege when I am chosen and trusted to interpret a weddingview profile
Specializing in lifestyle & weddings, Esther is a fine art photographer based in Southern California. She is recognized for her light-filled, soft,view profile
Hi! I'm Erin McGinn, a wedding and lifestyle photographer located in Rhode Island. My background as a wedding planner, blog and wedding magazine editor,view profile
For more than a decade, I have worked as a professional photographer. Having an education in photography and hands-on industry experience has uniquelyview profile


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The OtherMonthDayStyle property is used to set or return the style of the days in the calendar that are not part of the current month.
|Specifies the style to set. See the Style Control to see the possible styles and their values
|Specifies the value of the specified style
The following example shows one way to set the OtherMonthDayStyle in a calendar:
The following example shows another way to set the OtherMonthDayStyle in a calendar:
Set OtherMonthDayStyle of a Calendar control (with declaration and script)


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Soil & Groundwater Companies in Central African Republic
Category: Soil & Groundwater ×
Industry: Energy ×
Location: Africa ×
Location: Central African Republic ×
ICT International
based in Armidale, AUSTRALIA
ICT International has been designing, manufacturing and providing monitoring solutions for soil, plant and environmental research since 1982. The success of our instruments stems from our worldwide experience in the application of quantitative technologies to soil moisture monitoring, plant water use and environmental studies.Our systems are at the cutting edge of ...
based in Varennes, QUEBEC (CANADA)
Solmax is a worldwide leader in the manufacturing of PE geomembranes and offers both containment and fluid transportation solutions including HDPE pipes, valves, fittings and accessories. Manufactured from Canada, Malaysia and Chile, its products are sold in over 60 countries and are used by the biggest names in the mining, petroleum, waste management, water, and civil ...
Environmental Source Samplers, Inc.
based in Wilmington, NORTH CAROLINA (USA)
Environmental Source Samplers, Inc. (ESS) has a long and proven track record. We have been providing air quality related services since 1979. Headquartered in Wilmington, North Carolina, ESS has provided stack testing services in most of the fifty states and internationally. ESS serves clients in many industries including: Aluminum, Asphalt, Automobile, Carbon, ...
based in London, UNITED KINGDOM
Ecometrica is passionate about clear, correct, and meaningful environmental management that helps businesses meet their sustainability challenges, reduce costs, and increase overall performance. We help our clients measure their environmental impacts, from carbon, energy, paper, waste and water management all the way through to full supply chain accounting. We have global ...
Innov-X Canada
based in Vancouver, BRITISH COLUMBIA (CANADA)
Innov-X Canada is focused on the safe, responsible and effective utilization of XRF and XRD analytical equipment. In doing so Innov-X Canada brings a superior suite of non-destructive analyzers to market that provide qualitative and quantitative material characterization for detection, identification, quality control, site assessments, process control, regulatory ...
Perillon Software Inc.
based in Littleton, MASSACHUSETTS (USA)
Perillon Software delivers comprehensive cloud-based Operational Risk & Compliance Management solutions to mid to large-sized companies in energy, power, industrial, manufacturing and process industries. As the technology leader, Perillon helps Environmental, Health, Safety and Quality (EHS&Q) professionals at all levels in the organization measure, manage and analyze ...
ThermoEnergy Corporation
based in Worcester, MASSACHUSETTS (USA)
ThermoEnergy is an integrated technologies company engaged in the worldwide development and sales of sustainable technologies for wastewater recovery and reuse. The company's water division is a leading provider of custom designed, turn-key systems for wastewater treatment and reuse, chemical/metals recovery, and water conservation. ThemoEnergy`s CAST® and RCAST ...
Edinburgh Instruments Ltd
based in Livingston, UNITED KINGDOM
Edinburgh Sensors are proud to be global providers of high quality Gas Sensor solutions. Our diverse range of gas sensors use the latest technology to enable reliable, accurate and continuous gas detection. Established for over 20 years, Edinburgh Sensors use proven technology to deliver OEM Gas Sensors and Gas Monitors / Indoor Air Quality Monitors that are smart, ...
Geothermal Resources Council
based in Davis, CALIFORNIA (USA)
With the experience and dedication of its diverse, international membership bolstering a 40-year track record, the Geothermal Resources Council (GRC) is the world's premier geothermal association. The GRC serves as a focal point for continuing professional development for its members through its outreach, information transfer and education services.GRC GoalsThe GRC is a ...
Titan Cloud Software Company, LLC
based in Brentwood, TENNESSEE (USA)
Titan offers the petroleum industry`s most powerful cloud software--the solution used by the largest c-stores, commercial fleet, and petroleum marketers in the world. This software protects your valuable underground storage tank (UST) and above ground storage tank (AST) assets by enabling proactive management of all regulatory and operational risks. Its full array of ...


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Kraft Issues Voluntary Recall Of Some American Singles Cheese Product
Kraft Issues Voluntary Recall Of Some American Singles Cheese Product
Indie Horror Movie 'It Follows' Gets Wide Theater Release
Unlock your personal NFL Now stream by signing in to NFL.com
Kraft Foods Group said on Friday it is voluntarily recalling 7,691 cases of some varieties of its Kraft American Singles as a precautionary measure after a supplier failed to store an ingredient correctly. The Northfield, Illinois-based company said the recall affects four varieties of Kraft American Singles Pasteurized Prepared Cheese Product. The recall is for products with "Best When Used By" dates of Feb. 20, 2015, and Feb. 21, 2015. Kraft said that any of the product in question should not be consumed and should be returned to the store where purchased for an exchange or full refund.
Two weeks after opening in just four theaters, the indie horror movie "It Follows" is getting a wide release this weekend. The film has been getting positive reviews for its originality, paranoia-inducing scenes and lack of a typical villain. Actress Maika Monroe said in a recent interview, "The movie starts out with my character, Jay, and her boyfriend having this sexual encounter. She soon realizes that he has passed something on to her, this thing, whatever we want to call it, that's haunting her, and the only way to get rid of it is to pass it on."
Less than a week after leaving the Republican Party, Glenn Beck had something to say about how Ted Cruz should approach the primaries. Leave Scott Walker and Rand Paul alone. Otherwise, Beck warned, "we're just going to end up with Jeb Bush." Cruz professed his admiration for Paul and Cruz, but didn't quite commit to refrain from criticizing them. That's because he can't. The Republican presidential race can be split into the conservative primary and the establishment primary. Beck isn't wrong that a divided conservative vote could benefit Bush, but there also has to be a clear conservative candidate to challenge him. In most polls, Cruz is well behind Walker. He is also trailing Ben Carson, Mike Huckabee and Paul. Cruz obviously needs to change that dynamic.
Reynolds revealed the costume from Tim Miller's adaptation of the Marvel Entertainment character on Twitter Friday morning, with a twist on the classic Spider-Man motto. Previously, Reynolds had teased the mask and gloves of the outfit on Twitter, but this is the first time the full outfit is revealed in all its glory. It's impressively faithful to the comic book visual, making it perhaps the most faithful movie superhero outfit next to Spider-Man's — and even the movie versions of that outfit made some changes along the way. Deadpool, which will also star T.J. Miller, Gina Carano and Morena Baccarin, will be released Feb. 2016.
Senate lawmakers passed an amendment Thursday that would block the federal government from taxing carbon dioxide emissions, a popular idea among Democrats to fight global warming. Four Democrats joined Senate Republicans in backing the amendment to the 2016 budget bill introduced by Sen. Roy Blunt, a Missouri Republican. The amendment would create a reserve fund that would prevent the government from taxing carbon emissions from emitting entities, like power plants or refineries. "Once again, a majority of Congress has spoken out against a carbon tax," Oklahoma Sen. Jim Inhofe, chairman of the Environment and Public Works Committee, said in a statement after the vote.
Drum Roll Please, And today's good guy award goes to Tim Cook. Rich people often get rap for being greedy and awful, but clearly that's not always the case. Apple CEO Tim Cook has announced that he'll donate the lion's share of his money to charity after paying for his nephew's college education. On Cook's current stock portfolio he's worth $120 million, and that number jumps dramatically to nearly $700 million were it to be fully vested. Cook has publicly proclaimed his interest in issues like AIDS research, human rights and immigration reform, and says he's already donated millions albeit quietly. The places Cook in the same ranks of his Mega rich Contemporaries, Bill Gates and Warren Buffet who also intend to give away the bulk of their wealth by the time they pass. Frankly, this is the kind of behaviour should be more pervasive amongst the rich. Better health, better learning and a better quality for all life becomes a greater possibility when the uber caking return their wealth to the economy that gave it to them.
Sin City Saints Episode 1: The Fool Monty
Sin City Saints Episode 2: Smoke and Mirrors
Sin City Saints Episode 3: Gone Catfishing
Sin City Saints Episode 4: Mrs. Wu's Tang
Sin City Saints Episode 5: A Basket Full of Rainbows
Sin City Saints Episode 6: You Booze, You Lose
Andy Grammer LIVE Concert
Big Data LIVE Concert
Fifth Harmony - Worth It - Live from Wilbur Theatre #YahooLive
Ricky Martin - Livin La Vida Loca - Live from Gramercy #YahooLive
Flogging Molly - Drunken Lullabies - Live from Hollywood Palladium #YahooLive
Black Joe Lewis - Skulldiggin - Live from Brazos Hall #YahooLive
Which Rapper Has a Fever For More Will Ferrell Cowbell?
Fit It In: Never Skip This 2-Minute Warm-Up
Sounds That Have Emerged From Will Ferrell
Cote de Pablo Shares Things That Might Surprise You About 'The Dovekeepers'
Will Ferrell Brings Bird on 'Conan', Doesn't Want to Talk About It
The Pogue Review: Robo Vacuums


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Little did Nicholas Gurule know that when he launched the javelin on July 29, the object would slice out a piece of history.
Gurule, who competes as part of the Valley Track Club in nearby Villanueva, placed seventh in the turbo javelin throw, clearing a distance of 129 feet, 9 inches (39.56 meters) in the United States Track and Field National Junior Olympic Championships in Sacramento, Calif.
As one of the top eight throws among 11- and 12-year-old boys in the event, Gurule's performance earned the area native a USATF All-American honor.
With the recent meltdown of the Fiesta Council, it's clear that changes are needed. At a recent Las Vegas City Council meeting, Mayor Alfonso Ortiz suggested the possibility of the city taking over Fiestas de Las Vegas, held annually around the Fourth of July.
Las Vegas' traffic patterns could be changed dramatically if Highlands University makes National Avenue into a one-way street.
The Highlands Board of Regents last week approved a master plan that includes that change to National. It was part of a plan designed by Denver-based architectural firm StudioINSITE.
The state police have arrested two more repeat DWI offenders, reports state.
Alfred M. Ulibarri, 39, 1317 Keen St., was charged with aggravated DWI for his fourth offense, and Jose A. Mendoza, 40, of Los Lunas, was arrested for his third DWI, state police said.
Shortly before midnight July 22, a state police officer noticed Ulibarri on New Mexico Avenue speeding a few miles per hour over the limit, so the officer stopped him, according to a police report.
Owners Bob and Dolores Gerhardt of Las Vegas found themselves celebrating as First Down Dash colt He's Too Icy For Me picked up his first stakes win in the $625,000 Rainbow Futurity at Ruidoso Downs.
He's Too Icy For Me broke ninth but quickly made his way to second, then first to cross the wire a nose ahead of favorite American Runaway.
The favorite gave a good bid for the win but in the end was outnodded. Despite showery weather and a sloppy track, He's Too Icy For Me ran the 400 yards with a time of :19.409.
The state Transportation Department needs to improve its planning. The Associated Press reported that the state sold a piece of land next to an Albuquerque interchange for $306,000 in 2005, but bought it back for triple that price per square foot just four years later.
Even when the state sold the land, top officials were seeking money to expand the interchange. Those plans came to fruition, and the state needed the old land back. It was a bad deal for taxpayers.
Mayor Alfonso Ortiz said last week it's possible that the city could take over the operations of Fiestas de Las Vegas. He called it a "viable option."
For years, a nonprofit group known as the Fiesta Council has run the annual event, which is during the Fourth of July weekend.
Still, the city is heavily involved with the festivities. It sets up the dance floor and bleachers, readies the park and provides help with public works, solid waste, fire protection and law enforcement.
I was thinking about my co-workers the other day, and I realized something about them: None of them smoke.
At least that I know of.
After asking around, I found out that one of my co-workers smokes. But she apparently keeps the habit pretty hidden.
Even three years ago, I remember that a number of my co-workers used to puff their cigarettes out the back door. But they have either quit their habit or left the Optic.
Former New Mexico Highlands University coach Cindy Roybal is the recipient of this year's Ralph Bowyer Coaching for Character Award presented by the New Mexico High School Coaches Association.
The Las Vegas City Schools board met last week to face a budget reality: The state plans to further cut spending for schools and other agencies.
Now, East officials are mulling over how to deal with the drop in state revenue. They're even mentioning the possibilities of closing a school and cutting some athletic trips.
In the coming days and weeks, other area school districts and colleges and the state hospital will have to grapple with these cuts. The Albuquerque school district is contemplating more staff reductions.
Copyright © 2015
The Las Vegas Optic is your source for local news, sports, events, and information in San Miguel County and Las Vegas, NM, and the surrounding area.


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Okra and other vegetables in container
I am planning to grow okra in container. What size container do I need and how many plants can I have per container. Also, how many plants do I need to have to get enough okra for a family of 3?
I have a shady, east facing balcony. I hardly get 2 hrs of direct sun each day. What other vegetables can I grow in my balcony.
I like to use fabric gardening containers. They work really well with all types of vegetables and herbs, especially if you are growing them on your balcony.
Read this for more information on sizes and types:
Here is a link that might be useful: Gardening Containers
Srilu, I grew clemson spineless okra in 5 gallon pots last year, but it didn't really start to produce until I put it in direct sun against a south wall. I don't know how they will do on your balcony. They are some of the most heat-loving plants around. Perhaps you could arrange to grow some plants in pots in the parking lot of your building, or where you work?
Although there may be dwarf okra that you could grow in three gallon pots, I would not put more than one plant in a 3 or 5 gallon container and would have at least two plants per okra eater. Don't let the pods get over 3 or 4 inches before you harvest them or they will be woody and tough.
Good luck,
I agree about the shade, okra is a heat/sun loving plant, not going to do well with only 2 hours of direct sun. I'm in Florida and can grow it as a perennial, although it does slow down production from November through March. I've got three dwarf Baby Bubba plants in each 7 gallon container x 4 containers, that's enough to feed 2-3 people during the spring and summer. I honestly don't think you will do well though unless you can find a sunnier spot.


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Increasing Productivity and Reducing Operational Costs With Virtualization
Free registration required
Executive Summary
There is clear evidence that virtualization reduces capital expenditures, but what about operational cost savings? The presenter will share recent research findings and customer case studies that validate the opportunity to lower operating costs and transform how IT is managed with virtualization.
- Format: Webcast
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The Bernie Mac Show 2001 | TV Show Watchlist
The curmudgeonly comedian and his wife take in his sister's three young children in this Peabody Award-winning series. Mac's breakthrough was his appearance in Spike Lee's `The Original Kings of Comedy', in which he wowed audiences with his cutting humor.
Premiered: November 14, 2001
Rating: None
Season 5, Episode 18 What Would Jason Do
First Aired: March 31, 2006
Two boys want to take Vanessa to the senior prom, and Bernie wants to influence her decision. Trouble is, he knows she'll say no to whomever he prefers. Meanwhile, Bryana's soccer-field mishap makes her queen of the house, much to Jordan's chagrin.
Guest Cast
Naya Rivera Donna
Rodney J. Hobbs Rodney J. Hobbs Mr. Scott
Watchlist Sign In
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The Bernie Mac Show - Season 1
By 20th Century FoxBuy New: $9.99
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At Condor Ferries we believe that our people are our greatest asset and it is our commitment to deliver superior standards of service. From Accounting to Marketing, Engineering to Sales, there's an opportunity for everyone with Condor Ferries. Join us and begin the #GoodTimes.
We update this page as and when any positions become available. If you have any questions about career opportunities at Condor Ferries or you would like to send us your CV, please email us at firstname.lastname@example.com and a member of our Human Resources Team will come back to you as soon as possible.
Location: Poole
Fleet Engineering Intern
Placement to commence 1st of July 2015 Salary: To be advised
Placement Duration: 12 MonthsLocation: PooleRegion: Dorset
Disciplines accepted:A predicted 2:1 degree (minimum) in either a BEng, MEng or MSc - Mechanical - Naval Architecture / Marine
The role is an excellent opportunity for a motivated individual wishing to gain work experience in the marine industry. In addition to the technical analyst role, the chosen candidate will gain first-hand experience of working with and around experienced Marine Engineers, both in the Fleet Engineering office and in the field (through exposure to yearly ship dry-docks). This experience will provide a good foundation for any candidate interested in pursuing a career as a marine engineer or with a ship classification society.
Why a placement year with Condor Marine Services?
You will be employed as a full time Fleet Engineer and be treated as such.
Students aren't taken on as an extra resource; they are needed to fulfil the role of an engineer and contribute to the design and development of the vessels. The work is real, interesting and relevant to your studies.
Our open plan office layout and management strategy promotes effective and timely communication and teamwork.
As part of your responsibilities as a Fleet Engineering Intern you will be required to communicate with external suppliers as well as other departments within the company.
A mixture of office based analyst work and external visits to the vessels.
First Stage: Contact email@example.com with your CV and a cover letter outlining why you would like this opportunity and what you could bring to the role. A full job description is available upon request.
Second Stage: If successful, a company visit will be arranged followed by an interview with the Fleet Director.
Closing date for applications: 31 March 2015.
Email applications may be sent to: firstname.lastname@example.com
Closing date for applications is: 31/03/2015
Passenger Services Agent Seasonal - Part time
Condor Ferries 20 hours per week- shift work
Based in Poole Port
We are looking to recruit seasonal part time Passenger Services Agents to operate in our Poole Port, in the inaugural season of our new vessel "Liberation".
Main responsibilities of a Passenger Services Agent are:-
Key Skills & Personal Profile
Reporting to the local Passenger Services Management Team, you will be expected to work unsociable hours on a shift rota basis which will include weekends and bank holidays.
A full Job Description and remuneration details are available upon request.
To apply, please forward your CV and covering E Mail to; email@example.com
Closing date for applications is Monday 23 March 2015
Closing date for applications is: 23/03/2015


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Burrell High School Alumni
Lori Dodge
Class of 1985
Gary Torkeo
Class of 1977
Richard Brooks
Class of 1967
Fidelis Hendricks
Class of 1977
This site has been created for the former students of Burrell HS located in Lower Burrell, Pennsylvania. If you haven't already, we also encourage you to register for free on this site. You may also want to check out the latest news and school memories. If you are a Burrell alumn, we encourage you to register and be sure to sign up for the alumni newsletter.


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Top Global Market Stories
Europe Lower Ahead of Key UK Retail Sales Data
European indices are expected to open lower on Thursday following rough sessions in the US and Asia overnight. People have spent the last week trying to explain why US indices in particular have suffered over the last week, with some pointing to growing expectations of a summer rate hike and others highlighting the weakness in […]
Top Trade Ideas
Top trade idea for March 27th, 2015 – USD/MXN
Many traders will refuse an each way bet. That is, if a trade goes against them, they won't reverse and go in the new direction. Usually this is a result of at least one painful "whipsawing" as an inexperienced trader. Yet experienced traders know when to break the rules,, and the Mexican peso's positioning at […]
MT4 Trading & Education
How To Trade The Parabolic SAR in MT4
Hi, this is Shaun Overton with ForexNews.com and OneStepRemoved.com. In this 3 minute video, I'm going to introduce you to the Parabolic SAR indicator for MetaTrader 4. If you don't already have a demo account, you can follow along for free with an OANDA account by clicking the link below this video. Register For A […]
Featured Chart
Chart Of The Day For March 27th,2015 – EUR/USD
Talking Points:
- EUR/USD Technical Strategy: Flat
- Support: 1.0857, 1.0456, 1.0244
- Resistance:1.1051, 1.1263, 1.1513
The Euro rallied against the US Dollar as expected after putting in a bullish Piercing Line candlestick pattern. A daily close above the march 26 high at 1.1051 exposes the 38.2% Fibonacci retracement at 1.1263. Alternatively, a move below trend line resistance-turned-support at 1.0857 clears the way for a test of the 1.0456-541 area (March 16 low, the 23.6% Fib expansion).
Our long-term outlook calls for broad-based Euro weakness. With that in mind, we will treat any on-coming gains as corrective, looking to enter short at a more attractive level rather than a seeing the move higher as a buying opportunity. In the meantime, we remain flat.
Bitcoin Barely Changed, GBTC Begins Trading
Bitcoin is trading barely changed today, after the move higher faltered as well. The day started with a BTC rally from $243.38 to a high of $252.19, hit around mid-day. Prices didn't stay above $250 for long however and a slow and steady slide lower took BTC/USD back to the $243 mark. We are currently […]