SU434985A1 - DEVICE FOR PREPARATIVE SEPARATION OF HIGH-MOLECULAR BIOLOGICAL SUBSTANCES - Google Patents

Description

one

This invention relates to the preparative separation of high molecular weight biological substances, for example, nucleic acids, by gel electrophoresis.

A device for preparative separation of high molecular weight biological substances by gel electrophoresis is known, comprising upper and lower electrode chambers filled with a buffer solution with electrodes placed in them, communicating with the column in which the gel column is located, supported by a supporting filter, a membrane permeable to ions of the buffer solution, and a flow chamber for the buffer solution located in the cavity between the support filter and the membrane.

The supporting filter must be permeable to the molecules of the test substance and can be made in the form of a porous plate of glass or some synthetic material.

In order to increase the resolution of the device and obtain reproducible results in the proposed device, the supporting filter is made of batiste.

The drawing shows a schematic diagram of the proposed device.

The device contains the upper 1 and lower 2 parts of the column, connected by a threaded connecting sleeve 3. At the end of the upper part of the column, a supporting filter 4, made of batis, is fixed. To fasten the filter with a thread (rubber ring), an annular groove (not shown) is made on the outer generatrix of the upper part of the column. With

connecting the upper part of the column with the connecting sleeve 3, the filter 4 hall is located between the ends of the upper part of the column and the stop shoulder of the sleeve. Between the end of the lower part of the column and

the diaphragm 5 is clamped by the stop collar of the sleeve 3; it is made of cellophane or another material that is suitable for ions of the buffer solution. The lower part of the column has a channel 6 in its body, one end of which is connected by an opening 7 with the submembrane space of the lower part of the column, and at the other end of it a flexible tube 8 is made of fluoroplastic. Hole 7 is located in

close proximity to the membrane 5.

The filter 4 and the membrane 5 form a flow chamber 9 for the buffer solution in the column sleeve. Flexible tubes 10 and 11, made of fluoroplastic, are connected to the walls of the sleeve, communicating with chamber 9.

The upper part of the column is hermetically mounted in the connecting pipe of the bottom of the upper electrode chamber 12, in which the negative electrode 13 is fixed. The lower part of the column is fixed in the lower electrode chamber 14 so that the end of the lower part of the column is located from the bottom of chamber 14 at a distance equal to / 4 -7z height (depth) of the camera.

In the chamber 14 is fixed a positive electrode 15. The electrodes 13 and 15 are made of platinum.

The device works as follows.

Polyacrylamide gel is prepared by known methods, the concentration of which is selected depending on the substance to be tested.

Under the filter 4, a thin rubber film is placed between the upper part of the column and the supporting collar of the sleeve 3, after which the upper part of the column is filled with gel to a height of 20-30 mm. After gel polymerization, the top of the column is separated with sleeve 3, the rubber film is removed. Then the upper part of the column is reassembled in the sleeve 3. The gel column formed is retained by the filter.

The upper and lower electrode chambers and chamber 9 are filled with a buffer solution, using a pH 7.8 trisphosphate buffer, and known methods. Pre-electrophoresis is carried out to remove substances that absorb light from the ultraviolet region from the gel. After completion of the pre-electrophoresis, the upper and lower electrode chambers are filled with fresh buffer solution. The gel surface of the gel column, which is under a layer of buffer solution, is applied by known methods to the test substance.

One of the flexible tubes is connected to a known device, for example, a Mariotte flask, which creates a constant hydraulic nano buffer solution in chamber 9 at a constant flow rate of solution through this chamber within specified limits. Another flexible tube is connected to a known automatic collector and a substance fraction recorder, a Uwikord LKB type nanometer.

In electrode chambers, buffer solution circulates by known methods to preserve its composition. At the same time, in order to prevent the formation of air inclusions and to ensure the stability of the buffer solution along the RP and ionic strength in the sub-cavity of the lower part of the column, the buffer solution from the lower electrode chamber ii is discharged through channel 6 and tube 8.

The iia electrodes 13 and 15 provide a constant voltage that cuts the required current density of the electrophoresis. In the electrophoretic movement of the molecules of the analyte, the column of polyacrylamide gel plays the role of a molecular sieve, in connection with which

different molecules of matter penetrate chamber 9 but after different periods of time have elapsed from the beginning of the process.

Buffer solution passing through chamber 9 carries fractions of molecules of matter

in an automatic collector, b collector tubes collect individual fractions of molecules, the division into which for a particular type of test substance depends on the time of collection of the buffer solution in each

iroiirku, from the time of the farewell from the beginning of the process, as well as on the concentration of the polyacrylamide gel placed in the device column. The proposed device for separation

Substances such as nucleic acids, in particular RNA, allow identification and isolation of more than twenty separate fractions. The yield of the test substance exceeds 90%.

Pa using the same gel column when using idle electrophoresis in the forced mode in order to purify the gel from substance residues, the latter can be divided many times.

Subject invention

A device for the preparative separation of high molecular weight biological substances, such as nucleic acids, by gel electrophoresis, comprising upper and lower electrode chambers filled with a buffer solution with electrodes spaced in them and communicating with the column in which the gel column is located, which supports the supporting filter from the material permeable to the molecules of the test substance, the membrane, is impermeable to the ions of the buffer solution, and the flow chamber for the buffer solution, placed in

cavity between the supporting filter and the membrane, characterized in that, in order to increase the resolution of the device and obtain reproducible results, the supporting filter is made

from batiste.