Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
  • B01J20/04—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of alkali metals, alkaline earth metals or magnesium
  • B01J20/048—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of alkali metals, alkaline earth metals or magnesium containing phosphorus, e.g. phosphates, apatites, hydroxyapatites
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
  • B01J20/06—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
  • B01J20/28004—Sorbent size or size distribution, e.g. particle size
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
  • B01J20/282—Porous sorbents
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
  • B01J20/282—Porous sorbents
  • B01J20/283—Porous sorbents based on silica
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
  • B01J20/282—Porous sorbents
  • B01J20/284—Porous sorbents based on alumina
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
  • B01J20/282—Porous sorbents
  • B01J20/285—Porous sorbents based on polymers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
  • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
  • B01J20/287—Non-polar phases; Reversed phases
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/60—Construction of the column
  • G01N30/6004—Construction of the column end pieces
  • G01N30/603—Construction of the column end pieces retaining the stationary phase, e.g. Frits
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/60—Construction of the column
  • G01N30/6052—Construction of the column body
  • G01N30/6065—Construction of the column body with varying cross section
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/60—Construction of the column
  • G01N30/6091—Cartridges
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54386—Analytical elements
  • G01N33/54387—Immunochromatographic test strips
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/32—Bonded phase chromatography
  • B01D15/325—Reversed phase
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
  • B01D15/361—Ion-exchange
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
  • B01D15/3804—Affinity chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2220/00—Aspects relating to sorbent materials
  • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
  • B01J2220/64—In a syringe, pipette, e.g. tip or in a tube, e.g. test-tube or u-shape tube
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/02—Burettes; Pipettes
  • B01L3/0275—Interchangeable or disposable dispensing tips
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
  • G01N2030/8804—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 automated systems
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
  • G01N2035/1027—General features of the devices
  • G01N2035/1048—General features of the devices using the transfer device for another function
  • G01N2035/1055—General features of the devices using the transfer device for another function for immobilising reagents, e.g. dried reagents
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/26—Conditioning of the fluid carrier; Flow patterns
  • G01N30/28—Control of physical parameters of the fluid carrier
  • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient

Definitions

• the invention relates to a device and a method for separating and purifying molecules from a solution by adsorbing the molecules onto a matrix arranged in the device.
• Chromatography materials of various types have long been used in a tried and tested way for sample preparation in chemically, biochemically and molecular biologically oriented laboratories. Frequently, the samples are obtained on a microscale, in particular in the bio-chemical and molecular biological, but also in the analytical-chemical laboratory.
• the chromatographic materials are also used to extract certain components from the samples.
• the procedure to be referred to as solid phase extraction can also be used to clean and separate substance mixtures in solution.
• Removing and placing a storage container poses a risk of contamination for the employee, the environment and the sample itself.
• the danger exists particularly in the medical-technical field, for example when working with infectious material, or also in biochemical-molecular biological Laboratories, especially when handling radioactive substances.
• the object of the invention is therefore to provide a device which enables
• the object is achieved according to the invention by a device which is characterized in that the Matrix 2 in a truncated cone-shaped hollow body 1 open at both ends 4a, 4b, to which a cylindrical hollow body adjoins optionally at the further opening 4a, permeable to the solution between two
• Devices 3a, 3b is arranged, wherein the matrix 2 and the devices 3a, 3b together 5 to 50% of the
• Fill the volume of the hollow body and the further opening 4a of the frustoconical hollow body 1 can be connected to a pipette.
• Figures 1 and 2 show schematically the structure of a preferred embodiment of the device according to the invention.
• the matrix 2 is delimited by two devices 3a, 3b which simultaneously ensure that the matrix 2 is fixed in the selected position.
• the devices 3a, 3b are preferably clamped to the wall of the hollow body 1. However, in addition to fastening by tension, fastening by gluing the devices can also be achieved. Both methods lead to an adequate seal between the devices 3a, 3b and the wall of the hollow body 1.
• the further opening 4a of the two openings 4a and 4b is designed such that they fit on a commercially available pipette.
• a preferred embodiment of the device according to the invention consists in that the hollow body 1 is designed in the form of a pipette tip commercially available from specialist dealers (Brand, Gilson, Eppendorf).
• the matrix 2 of the device according to the invention preferably consists of a porous chromatography material, in particular based on silica gel, aluminum oxide, titanium dioxide, hydroxylapatite, dextran, agarose, acrylamide, polystyrene, polyvinyl alcohol . or other organic polymers, derivatives or from copolymers of the abovementioned carrier materials.
• the material forming the matrix 2 can also be a surface-modified, porous chromatography material based on silica gel, aluminum oxide, titanium dioxide,
• Hydroxyapatite dextran, agarose, acrylic acid, polystyrene, polyvinyl alcohol or other organic polymers, derivatives or from copolymers of the abovementioned carrier materials.
• ion exchange materials can be used as matrix 2 of the device according to the invention for the separation and purification of polar molecules.
• Further preferred embodiments of the device according to the invention can contain a reversed-phase and / or an affinity chromatography material as matrix 2.
• the pore size of the porous chromatography materials is preferably 20 to 1000 nm, and the grain size of the materials in particular 10 to 2000 ⁇ m, preferably 75 ⁇ m to 125 ⁇ m.
• the devices 3a, 3b fixing the matrix 2 are preferably designed as porous frits with pore sizes of 10 ⁇ m to 1 mm, preferably 70 ⁇ m to 2000 ⁇ m.
• the porous frits can consist of plastics, in particular Teflon (PTFE), polyethylene, polypropylene, polystyrene, polyurethane etc.
• PTFE Teflon
• inorganic materials such as glass and / or sintered metal are also suitable materials for the production of the porous frits.
• FIGS. 3a and 3b show a further embodiment of the device according to the invention, the simple manufacture of which is particularly advantageous.
• a removable cartridge 5 is inserted into the hollow body 1 and contains the matrix 2 between the devices 3a and 3b fixing the matrix.
• the device 3b is arranged above a net-like carrier 6, which preferably consists of the same material as the removable cartridge 5.
• the net-like carrier 6 and the cartridge 5 are produced in one operation, for example in the injection molding process, if cartridges are made of plastic, in particular polypropylene, the device 3a is arranged above the matrix 2 which in turn is arranged a porous, mesh-like cover 7.
• FIG. 3b shows the overall arrangement, consisting of hollow body 1 and removable cartridge 5.
• This overall arrangement, consisting of cartridge 5, devices 3a, b and cover 7 fixing the matrix 2 can be combined in a particularly advantageous manner with commercially available pipette tips become. In the simplest case, this is done by pushing the removable cartridge 5 into the corresponding hollow body 1, in particular a pipette tip.
• the cartridge 5 is dimensioned so that it exerts a slight pressure on the inner walls of the pipette tip in order not to be pushed up when absorbing liquid or a sample or to provide the necessary seal to the inner wall of the hollow body 1, preferably a pipette tip, and the
• the wall thickness of the removable cartridge 5 of the mesh-like carrier 6 and of the porous mesh-like cover 7 is matched to the pipette tip used.
• the advantage of the device according to the invention lies in the practical handling and the type of packing of the matrix 2, which allows the solution containing the molecules to flow in the back and forth direction.
• the frustoconical hollow body 1 is designed, for example, as a pipette tip, the pipette tip is immersed in the sample, the sample in the pipette tip is sucked up through the matrix 2 and then pressed out. This results in a higher efficiency of the adsorption, since the sample passes through the chromatographic material twice. Since it is possible to work with a closed system, contamination of the sample or danger to the environment is avoided.
• the device according to the invention simplifies the overall method of operation which is to be used in the separation and purification of molecules.
• biopolymers in particular nucleic acids and proteins, can also be fractionated quickly, easily and reliably.
• the method according to the invention is carried out in a special embodiment with a hollow body 1 designed as a pipette tip.
• the molecules containing, separating and cleaning the molecules can then be conveyed through the matrix 2 by means of a pipette. If the pipette tip is to be used as a column replacement in conventional processes, then - 1 -
• the pipette tip can also be connected to a disposable syringe using a silicone tube.
• the method according to the invention is suitable, in particular, for separating and purifying biopolymers such as nucleic acids and proteins by using ion exchange chromatography materials as proposed in German patent application P 36 39 949.3 as the matrix 2. It is silica gel-based chromatography material modified on the surface with anion exchange groups. If, for example, the matrix 2 is in a pipette tip, the nucleic acid is sucked through the matrix 2 with the pipette. The long-chain nucleic acids are adsorbed using buffers of low ionic strength. If you then want to utilize the short-chain nucleic acids, you push the solution back through the matrix 2 and catch it. Then further procedural steps can follow.
• the long-chain nucleic acids can be eluted again by elution with buffers of high salt concentration (high ionic strength) and processed accordingly.
• FIG. 4 shows an elution profile which can be obtained when anion exchange materials are used when using the device according to the invention. It has been shown that when using the material proposed for the separation of nucleic acids in German patent application P 36 39 949.3, an elution profile is obtained by means of which nucleic acids with increasing chain length can be eluted with increasing ionic strength. Thus, double-stranded DNA with base pairs> 500 is only obtained with an ionic strength of 1.3 M sodium chloride, while single-stranded DNA of the phage M 13 is eluted at 1.1 M sodium chloride. The elution peaks are so sharp that even "baseline" separations are possible. At an average ionic strength between 0.5 and 1 M sodium chloride, nucleic acids such as tRNA, 5 RNA and mRNA elute. At low ionic strengths of 0.1 to 0.5, polysaccharides,
• Proteins such as BSA (bovine serum albumin) and smaller ones
• a variant of the method consists in using an affinity adsorbent as matrix 2, as is proposed, for example, in German patent application P 36 27 063.
• the procedure is carried out analogously to the procedure described above.
• the method according to the invention ensures the following advantages, in particular in molecular biological operations:
• the purity in routine preparations obtainable when using the device according to the invention is at least comparable to samples obtained by Caesiu chloride density gradient centrifugation.
• the isolated nucleic acids are free of proteins, polysaccharides and other cell metabolites and show no inhibition of enzymes in enzymatic reactions.
• the device according to the invention can be used for the isolation and purification of nucleic acids from oligonucleotides to plasmids, the time expenditure being restricted to fractions in comparison to other processes.
• the device according to the invention is suitable for use in a separation and purification process for molecules, preferably biopolymers such as proteins and / or nucleic acids, in particular of other cell components.
• the use of the device according to the invention in various processes is explained in more detail in the following examples.
• the device according to the invention is used in a preferred embodiment, the pipette tip.
• a 70 ⁇ polyethylene frit with a 4 mm diameter and a thickness of 1.6 mm is introduced into a 1 ml pipette tip, suitable for Gilson Pipetan, and clamped by pressing. Then about 70 mg of the chromatographic material, proposed in German patent application P 36 39 949, are filled in dry. The chromatographic material is closed with a 5.7 mm diameter frit as described above and the frit is fixed in place by pressing. The procedure is analogous for 200 ⁇ l pipette tips or 5000 ⁇ l pipette tips.
• the pipette tip is equilibrated once with 100 ⁇ l of a suitable adsorption buffer. With a sample volume of 100 to 200 ⁇ l to be processed, it is sufficient to push the sample through the chromatography material about five times. However, if a larger amount of liquid is to be processed, such as is obtained after gel elution, the sample can be drawn through the matrix using a silicone tube that connects the pipette tip and a disposable syringe. It is then sufficient for the sample to pass the chromatography material only twice. The flow rate of the solution should not be higher than 1 ml per minute.
• Buffer A can be used as equilibration and adsorption buffer. Buffer A:
• buffers B, C and D are typically used as washing buffers.
• Buffer B Buffer C:
• An Eppendorf tube is filled with 1 ml of washing buffer and 150 ⁇ l are rinsed three times through the chromatography material in order to remove impurities. This step is repeated.
• the adsorbed nucleic acids are eluted either with an Eppendorf or Gilson pipette or with a disposable syringe, care being taken that the pipette tip is connected to the disposable syringe by means of a silicone tube. It simply becomes the elution buffer E or F
• Buffer E Buffer F:
• a pipette tip, produced according to Example 1, is hydrated with 50 mM sodium phosphate buffer and equilibrated by passing 750 ⁇ l buffer several times through the
• Pipette sucks.
• a plasmid DNA sample is typically obtained in a so-called mini-preparation by the following steps:
• the bacterial cells are incubated overnight and harvested the next day by centrifugation.
• the pellet is disrupted in 85 ⁇ l of an ice-cold solution of 50 mM Tris-HCl, pH 7.4, with 2 mg / ml lysozyme (freshly prepared) and incubated on ice for 10 minutes.
• 20 ⁇ l of a 0.5 M EDTA solution are added and incubated for a further 10 minutes.
• After adding 4 ⁇ l of 2% Triton X '100 the mixture is incubated on ice for a further hour.
• the sample is centrifuged in a laboratory centrifuge for 30 minutes at maximum speed, and 100 ⁇ l of the cell lysate freed from cell debris are transferred to another Eppendorf tube, after which 1 volume of 2 M sodium chloride, 100 mM MOPS, pH 7 and 5 ⁇ l Isoa yl alcohol can be added.
• a pipette tip according to the invention from Gilson, Brand, Eppendorf, manufactured according to Example 1, is equilibrated with 100 ⁇ l buffer C. Then 100 ⁇ l of an E. coli plasmid DNA sample is adsorbed on the chromatography material by pipetting the sample up and down five times.
• RNA extract is precipitated with ethanol and the pellet obtained is resuspended in TE buffer (10 mM Tris / 1 mM EDTA, pH 7.5).
• TE buffer 10 mM Tris / 1 mM EDTA, pH 7.5.
• the adsorption conditions are adjusted by adding 0.1 part by volume (based on the re-suspended solution) of 5 M sodium chloride and 0.2 part by volume of 250 mM MOPS, pH 7.0.
• the pipette tip is equilibrated with 1 ⁇ l buffer A.
• the sample is then adsorbed onto the chromatography material by pipetting up and down five times. One rinses with buffer A in order to eliminate impurities. Elution is carried out with 300 ⁇ l of buffer C.
• the RNA is precipitated by adding 2.5 parts by volume of ethanol. After standing at -20 ° C. for 30 minutes, centrifugation in an Eppendorf centrifuge takes a total of 30 minutes at the highest speed. The pellet
• the labeling reaction of the DNA is terminated by adding 2 ⁇ l 0.5 M EDTA per 20 ⁇ l sample volume, and the adsorption conditions are adjusted by adding 1 volume part of the buffer C.
• the total final salt concentration should be approximately 500 mM.
• the pipette tip according to the invention is equilibrated with 100 ⁇ l buffer B.
• the sample is adsorbed onto the chromatography material by suction up five times and squeezed out and washed with a total of 10 ml of buffer B in order to separate the unreacted nucleotides.
• the flow rate of the washing buffer should preferably be approximately 5 ml per minute.
• the labeled DNA is then eluted with a total of 300 ⁇ l of the buffer F.
• the sample can be used directly if it is diluted at least 1:20 with the buffer intended for hybridization. Otherwise the sample is precipitated and dissolved in TE buffer.
• the removal of a DNA linker from a DNA with more than approx. 400 base pairs in a cloning experiment is carried out as follows:
• the sample to be cleaned is diluted with 1 part by volume of the buffer C so that the final concentration of salt is approximately 500 mM.
• the pipette tip according to the invention is equilibrated with 100 ⁇ l of buffer A.
• the sample is adsorbed as in the previous examples.
• the pipette tip according to the invention is rinsed with buffer B in order to remove the contaminants.
• the DNA is desorbed and eluted with a total of 300 ⁇ l of buffer F.
• DNA is isolated by isopropanol precipitation (addition of 1 part by volume) and left on ice for 15 minutes, after which the sample is centrifuged in an Eppendorf centrifuge for the Duration of 30 minutes. Then wash carefully with 70% ethanol.
• the underlying reaction volume is 50 ⁇ l and contains no more than 100 mM sodium chloride. 5 ⁇ l of 0.5 M EDTA, pH 8.0, are added to the reaction volume in order to terminate the modification reaction. A volume part of the buffer C is added in order to set the adsorption conditions with a salt concentration of about 500 mM.
• the pipette tip according to the invention is equilibrated, the sample is adsorbed and then eluted as described in Example 8. The further treatment of the sample is also carried out as described in Example 8.
• the device according to the invention can also be used to efficiently separate agarose and acrylamide impurities. This is particularly necessary when gel elution of the sample has taken place.
• the DNA is concentrated at the same time, even if the starting volume is a few ml.
• the DNA has a size of> 400 base pairs and can be present in an amount from 5 ng to 15 ⁇ g.
• the sample preparation and the equilibration of the pipette tip according to the invention is carried out as described in Example 8.
• the sample is then transferred to a disposable syringe and pressed twice through the pipette tip according to the invention.
• the flow rate should be around 250 ⁇ l / in.
• the pipette tip according to the invention is then washed with 5 ml of buffer B at a flow rate of 5 ml / min.
• the further treatment of the DNA sample is carried out as described in Example 8.

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• 1987
  • 1987-05-22 DE DE19873717211 patent/DE3717211A1/de active Granted
• 1988
  • 1988-05-19 WO PCT/EP1988/000442 patent/WO1988009201A1/de unknown

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