WO2011117307A2

WO2011117307A2 - Method, reagents, and device for producing an analytical gel

Info

Publication number: WO2011117307A2
Application number: PCT/EP2011/054468
Authority: WO
Inventors: Hans Robert Attig; Rainer Dahlke; Andy Wende
Original assignee: Qiagen Gmbh
Priority date: 2010-03-26
Filing date: 2011-03-23
Publication date: 2011-09-29
Also published as: WO2011117307A3; DE102010003311A1

Abstract

The invention relates to a method and device for producing an analytical gel, such as an agarose gel or a polyacrylamide gel. The invention further relates to reagents for carrying out the method. The reagents and/or substances required for the production, hereinafter referred to as reagents, are poured into one or more areas intended therefor in a gel preparation device, in metered form, with the exception of water, for example in the form of a tablet or in packaged form. A chamber of the gel preparation device is completely or partially filled with a suitable quantity of water, typically 50 to 1000 ml. In addition, a metered form of the reagents, thus for example a tablet, is introduced into the chamber. The water is heated and the solution is subsequently cooled.

Description

Process, reagents and apparatus for the preparation of an analytical

gels

The invention relates to a method and a device for producing an analytical gel such as an agarose gel or a

Polyacrylamide gels. The invention further relates to reagents for carrying out the method.

Analytical gels and processes for the preparation are described in the document "Benchgulde" from Qiagen GmbH in Hilden, which can be downloaded in digital form over the Internet The publication JP 1 221 651 A discloses another method for producing an analytical gel An agarose gel is a gel used in agarose gel electrophoresis. To prepare the gel, agarose is boiled up in a buffer and

then cooled. The buffer in the case of a TAE agarose gel includes water, tris (tris (hydroxymethyl) aminomethane), glacial acetic acid and EDTA

(Ethylenediaminetetraacetic acid). When a TBE agarose gel is prepared, the buffer comprises water, tris, boric acid and EDTA.

Regularly, agarose gel is made manually in a laboratory just prior to its use because the shelf life of an agarose gel is limited to a few weeks.

For the manual preparation of the agarose gel In a laboratory, a

Fine balance required to weigh the amount of agarose required, a microwave for boiling the buffer with agarose therein, a vessel for making buffer, a vessel into which the desired amount of buffer is filled and in which the buffer is boiled together with the agarose. and a stirrer to stir during cooking. For manual production in a laboratory, it takes about 10 minutes to weigh and boil agarose, and another 10 minutes to cool the gel down to casting temperature. It is an object of the invention to develop the production of an analytical gel welter.

The object is achieved by a method having the features of claim 1. Moreover, a first additional claim relates to a metered form of required reagents for carrying out the method. A device provided for carrying out the method comprises the features of a sibling apparatus claim. Advantageous embodiments emerge from the subclaims.

To achieve the object, claim 1 relates to a method for producing an analytical gel in a gel preparation device. In the

Gel preparation devices are needed for the preparation

Reagents and / or substances - hereinafter referred to as reagents - with the exception of water In dosed form, such as in the form of a tablet or, for example, in packaged form, filled in one or more designated areas. A chamber of

Gel preparation device is filled in whole or in part with an appropriate amount of water of, for example, up to 1,000 ml. However, typically 1,0 to 250 ml. The water comes either from an external one

Water source or from a reservoir of

Gel preparation device in which water has been previously filled.

In addition, a metered form of the reagents, so for example a tablet is brought into the chamber. Are the needed

Reagents have been dosed through a package, so the package can be flushed with the water so that the contents of a package so enters the chamber. The total weight of the reagents usually exceeds 20 grams, especially 1 0 grams not. If a smaller amount of agarose gel is to be prepared, then the total weight of the reagents is usually up to 5 grams. A feeder a metered form of reagents may be prior to the introduction of water into the chamber, thereafter or during the introduction of water. Thus, in the case of a tablet, it is preferably placed in the chamber before the chamber is filled with water. Subsequently, the contents of the

The chamber is heated with heating means of the gel coating device so that a solution forms from the water and the reagents, from which the desired gel is formed by cooling. The solution thus prepared is passed out of the gelation device. The process can be carried out in a laboratory automated, without having to provide a complex apparatus. In contrast to a manual production In a laboratory, an automated and accurate preparation of analytical gels is possible. An important feature of the prior art is that one for a

Gel preparation required amount and composition of reagents In a metered form. The dosed form of needed

Reagents allow the preparation of the desired gel by dissolving in a designated amount of heated water and then cooling. The required reagents are thus brought into the Gelzuelteltungsvorrlchtung already dosed state. In the laboratory, therefore, the various required reagents do not have to be dosed individually, which is able to reduce the tent expenditure and possible preparation errors considerably. All reagents are in particular in the form of solid and / or as a finished mixture in order to minimize the space requirement.

If the reagents are in dried form, ie as a solid, they are also essential in comparison to precast gels

shelf-stable.

A metered form is sized so that it can be used to prepare an analytical gel volume for laboratory use. Thus, from a metered form, such a quantity of gel can not be produced which corresponds to an industrial production in order to carry out analyzes in many different laboratories with the gel produced. In particular, can be from a Make a dosed gel form with a maximum volume of one pint, preferably 250 ml.

If the reagents required for a gel preparation are contained in a packaging, then the reagents are preferably packaged airtight through the packaging in order to optimize shelf life. The contents of a package are then heated in water and preferably

additionally dissolved by stirring. After cooling, the desired analytical gel is then present without further steps having to be carried out. In particular, no further reagents are required

to be added.

The reagents are present in a packaging, in particular in pulverulent form, in order to be able to bring them easily and quickly into the chamber, in particular by introducing water into the packaging. The water flowing out of the packaging then transports the contents of the package into the chamber, which is then filled with water and reagents at the same time.

The gel preparation device does not have to be complicated

Dosing devices have to mix reagents in a suitable amount and composition with a suitable amount of water. The gel preparation device may be adapted to receive a singular or plurality of metered forms of the required reagents, eg, a plurality of tablets or a plurality of packages. Each individual tablet then already comprises all the reagents required for the preparation of the desired gel in the desired amount. Such a tablet must therefore only in a suitable, heated amount of water within the

Gel preparation device to be solved. The same applies to the contents of a packaging. Each package contains the required reagents In required quantity and composition In ready-mixed form. In one embodiment of the method is the chamber with a

Provided stirring device. When water and reagents, such as a tablet or the contents of a package, have been brought into the chamber, they are agitated by means of the agitator to aid in dissolving the reagents in the water. In particular, it is stirred during and / or after the heating.

To dissolve the reagents in the water, the water is preferably heated to a temperature of at least 80 ° C. In the case of TBE agarose gels, this temperature has been found to be sufficient to prepare from the contents of a dosed form and the water a solution from which the desired gel is formed by cooling. The water is in an embodiment with a hotplate of the

Gel preparation device heated. But it can also be used other heating means to the contents of the chamber to the desired

Temperature to warm. To control the temperature in the chamber, this is in one

Embodiment provided with a sensor, the temperature of the

Chamber and / or the temperature inside the chamber is able to measure. In one embodiment, the gel preparation device comprises a water tank, ie a reservoir for water, in order to be able to operate the gel preparation device independently of a water connection. The Gelzuveteltungsvorrlchtung can also with an external

Water connection can be connected to it to initiate water into the chamber in due course.

The gel preparation device may include a flow meter to allow the chamber to be filled variably with a desired amount of water automatically, thereby covering gels of different volumes. However, the gel preparation device can also be such that the volume of the chamber is always completely filled with water so as to be able to provide a defined amount of water for gelatinization. A metering device, with which a desired volume of water is provided in the chamber, can then be omitted, which simplifies the structure of the gel preparation device.

In one embodiment of the invention, the prepared solution is cooled in the gel preparation device, preferably to less than 60 ° C and preferably with an active and / or passive

Kühlelnrichtung. In one embodiment, the

Gelzuelteltvorrlchtung as an active Kühlelnrichtung a Peltler element with which the prepared solution is cooled before this from the

Gel preparation device is passed out. Alternatively or additionally, the device comprises, for example, a spiral,

Spiral, in particular made of metal existing pipe as passive

Cooling direction, over which the prepared solution from the

Gel preparation device is passed out and in which this solution is then passively cooled due to the relatively cool ambient temperature then to the extent desired. The pipe or one

comparable passive cooling device represents a very big, good

heat-conductive surface ready, over the comparatively fast heat with the environment, in which usually room temperature prevails,

can be exchanged. By means of an active and / or passive

Cooling the cooling device accelerates the cooling of the solution and thus providing the desired analytical gel.

In one embodiment of the invention, the solution prepared from reagents and water is poured out of the gel preparation device into a

Capillary pressed in. The gel can be hardened in the capillary and stored until it is used. Converting a prepared gel directly into caplets increases the shelf life. Alternatively, the prepared gel can be transferred to a gel pouring cup, which is in the Usually short term is used to perform the desired analysis using the analytical gel. The desired analysis is always performed in the same place. The gel produced is thus not first transported from an industrial production site to a laboratory in order to carry out an analysis in the laboratory with the aid of the gel.

In order to be able to push out of the gel preparation device the solution prepared in the gel preparation device and preferably subsequently cooled with the aid of cooling devices, the device comprises

Gel preparation device in one embodiment, a pressing means such as a compressor or a pump, with the prepared solution, for example via a filter and / or a nozzle from the

Gel preparation device can be pressed out. At this

For example, the prepared solution may be readily urged into a capillary, for example, which will be immediately used for analysis in due course.

In one embodiment of the invention, the

Gel preparation device a filter through which the prepared solution is pressed through. The filter is used to hold back impurities and / or to separate any air bubbles from the gel. The filter may have, for example, 100 μπη to 1 mm, preferably up to 400 μπη large pores and, for example, consist of metal.

In one embodiment, a metered form of the reagents comprises one or more information about the content and composition of the metered form and / or instructions for the preparation, preferably at least in machine readable form, such as in printed form as a barcode or stored on a RFID CPZ. The gel preparation device then preferably comprises means for a

Read out the machine readable information to then automatically perform the preparation depending on the read-out cooking instructions. For example, a package with the therein Reagents then contains instructions on how to at least heat up the amount of water to which temperature. The

Gelberbelungsvorrtchtung then provides the required amount of water in the automated chamber, and it is automatically heated to the predetermined temperature, without a user would have to make a setting.

The Gelzuveteltungsvorrtcht Is basically set up

programmed or programmable that this is able to produce and output a gel as described automated.

The invention reduces the number of steps required to produce an analytical gel in a laboratory, ie a gel with which an analysis is subsequently carried out. The risk of injury from boiling up In the lab is minimized. A misdosing is avoided by the invention.

Figure 1 shows an external structure of a gel preparation device.

FIG. 2 outlines an internal structure of a gel covering device.

1 shows a Gelberbereltungsvorrlchtung 1 with an area 2 for a water supply and metering unit. At the front there is a

Control panel 3, via which the Gelzubereltungsvorrlchtung 1 operated, so for example, in progress and can be stopped. The front comprises a drawer 4, in which a tablet or cartridge 5 can be inserted. About a tempering unit and nozzle 6 is prepared from water and the tablet or the contents of the cartridge gel from the

Gelbereltungsvorrlchtung 1 pressed out. Below the tempering unit and nozzle 6, there is a free space to place a container here.

FIG. 2 shows the internal structure of a gel-covering device. From a water tank 21 by means of a pump 22 water In a heating area 23 is heated. From the heating area 23, the heated bzwerhitzte water is introduced into a chamber 24. The chamber 24 comprises an insert 25, in which the required reagents are, for example, in the form of a tablet. The gel prepared in the chamber 24 is passed out of the gel preparation device through a cooling region 26. It thus enters into a gel collecting container 27.

Hereinafter, compositions are named from which a desired amount of gel can be prepared. So will in the

The following table indicates which buffer component in which amount must be provided in dried form in order to prepare desired amounts of gel. In addition, the tables indicate which amount of water to use to get out of the

Solid mixtures to produce the respective gel. These are typical examples. The compositions may therefore also be chosen differently in order to produce gels therefrom.

TAE gels with different agarose concentrations and total volumes

ΤΒΕ-ΘΘΙΘ with different agarose concentrations and total volumes

Urea agarose gels

In the case of urea agarose gels, urea is additionally added in the following ratio on the basis of the TAE or TBE gels.

In the following, common agarose concentrations are listed in TAE or TBE gels and the amounts of agarose required for the preparation are given for each gel size;

Example of use:

For a 100 ml TAE gel with an agarose concentration of 1%, 0.725 g Trls acetate, 0.037 g EDTA and 1, 0 g agarose are combined as a solid, for example as a tablet and in the Gelläßvorrlchtung with 1 00 ml of water.

Claims

claims

1. Process for the preparation of an analytical gel In a

Gel preparation device with the steps:

Filling of reagents required for the preparation of an analytical gel in metered form into a gel preparation device (1),

Introducing water into a chamber (24) of the

Gelzubereitungsvorrichtung,

Adding the contents of a metered form to the gelation device chamber (24);

Heating the water in the chamber with heating means (23) of the gel coating device (1) for the preparation of a solution of the water and the reagents,

Passing the prepared solution from the

Gelberberungsungsvorrtchtung (1).

2. The method according to claim 1, characterized in that the

Gelberbelungsvorrlchtung (1) comprises a stirring means, me the introduced into the chamber, heated water and the reagents therein is stirred.

3. The method according to any one of the preceding claims, wherein the water is heated to at least 80 ° C.

4. The method according to any one of the preceding claims, wherein the water from a water reservoir (21) of the

Gelberberungsungsvorrlchtung into the chamber (24) is initiated.

5. The method according to any one of the preceding claims, wherein the solution prepared from the reagents and water in the

Gelzuveteltvorrlchtung means of an active and / or passive cooling device (26) is cooled.

6. The method according to any one of the preceding claims, wherein the solution prepared from the reagents and water from the

Gelberbelungsvorrlchtung out into a capillary is pressed into.

7. The method according to any one of the preceding claims, wherein the prepared solution by means of a pressing device (22) of the

Gelbereltungsvorrlchtung is pushed out of the Gelzuveteltungsvorrlchtung out.

8. Composition of reagents from which an analytical gel can be prepared by heating with water, thereby

in that the composition is present or packaged as a tablet.

Composition according to the preceding claim, consisting of agarose, tris-acetate, and EDTA or trls, boric acid EDTA, agarose.

10. Composition according to the preceding claim, characterized

characterized in that the composition additionally contains urea.

11. Composition according to one of the two preceding claims, characterized in that the total weight of

Composition is not more than 20 grams, preferably not more than 1 0 grams.

12. The composition according to any one of the three preceding claims, characterized in that all reagents are present as a solid.

13. gel preparation device (1) for carrying out a method according to one of the preceding claims 1 to 7 with a chamber (24) and with means (21, 22) for the introduction of water into the chamber (24) of Gelzuveteltungsvorrlchtung,

with means (25) for receiving a package or a tablet, with means for delivering the tablet or the contents of the tablet

Packaging In the chamber,

with heating means (23) for heating water in the chamber to a temperature of 80 to 1 00 ° C,

with a cooling device (26) for cooling a solution prepared in the chamber (24),

with means (22) for discharging a solution prepared in the chamber from the gel preparation device.

A gel-sealing device according to the preceding claim, comprising a filter via which a solution prepared in the chamber (24) can be passed out of the gel-contacting device (1).

15. Gelberbereltungsvorrlchtung according to one of the two preceding claims with a Peltler element and or a spiral or helical or multiple reciprocating metallic tube as a cooling device.

16. Gelberelteltvorrlchtung according to one of the three preceding claims with a metering device for the introduction of a

given amount of water into the chamber.