JPS63222254A - Recovery cell for electrophoresis apparatus - Google Patents

Abstract

PURPOSE:To prevent the mixing of an inhibitor, by providing a recovery chamber for recovery of a transmission charged high polymer adjoining a gel chamber of an electrolysis apparatus by way of a glass filter penetrating to an opposed electrode through a cellophane film. CONSTITUTION:An electrophoretic tank 10 of a horizontal type electrophoresis apparatus filled with an electrophoretic buffer 11 is prepared and a recovery cell 13 is set in a gel chamber 1 to recover a gel 12 containing a desired charged high polymer. Power is fed with a negative pole on the side of the gel chamber 1 and with a positive pole on the side of a recovery chamber 4 to perform electrophoresis. A negatively charged high polymer moves to the recovery chamber 4 in the gel to be shifted to the recovery chamber 4 passing through a glass filter 2. A cellophane film 3 located on the positive pole side of the recovery chamber 4 will not let the charged high polymer pass, hence having the charged high polymer staying thereon 3. Fine gel fragments moving together with the charged high polymer are left in the gel chamber 1, refused to pass through the glass filter 2. Upon the end of the electrophoresis, the charged high polymer on the cellophane film is separated from the cellophane film by reversing current to electrically energize in a short time and taken out together with the buffer at a recovery takeoff portion 14.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a recovery cell used in an electrophoresis device, and more specifically, to a recovery cell used in electrophoretically separating cargo polymers such as DNA. The present invention relates to a recovery cell for an electrophoresis device.

[Conventional technology]

Separating charged polymers such as DNA by electrophoresis is used in basic genetic engineering operations to extract specific fragments (specific genes) from DNA fragments cut with restriction enzymes. .

In this operation, DNA fragments are separated by electrophoresis using an agarose or polyacrylamide gel as a carrier.
The DNA fragments are developed depending on their length, and the target DNA is recovered from the corresponding gel as an aqueous solution. This recovery can be carried out, for example, by cutting out a gel block containing the desired DNA fragment, sealing the block in a dialysis tinib with a small amount of electrophoresis buffer, and immersing the sealed block in the buffer of a horizontal electrophoresis tank. The charged DNA is electrically transferred from the gel to a buffer through electrophoresis, and the buffer containing the DNA is removed to recover the DNA.

[Problem that the invention seeks to solve]

In DNA recovery using dialysis tubing as described above, especially when agarose gel is used, minute fragments of the gel are mixed into the recovered DNA during the recovery operation, and this becomes an inhibitor of the restriction enzyme. Since the DNA cannot be removed by simple purification, there is a risk that the recovered DNA will not be able to be used effectively. In addition, the DNA recovery rate is not necessarily good, with only 10%
There are some very bad things like: Furthermore, the gel to be handled is very fragile and the dialysis tubing that houses the gel is soft, so the operation is complicated and requires care.

This invention was made based on the above-mentioned background, and its purpose is to prevent the contamination of inhibitory substances such as gel minute fragments and recover charged polymers such as DNA with easy operation and high yield. An object of the present invention is to provide a collection cell for an electrophoresis device that can be used for electrophoresis.

[Means for solving problems]

The present inventor has conducted various studies on a means for collecting charged polymers using an electrophoresis apparatus, and has found that the above object can be achieved by the collection cell for an electrophoresis apparatus according to the present invention. That is, the collection cell for an electrophoresis apparatus of the present invention has a gel chamber which communicates with one electrode of the electrophoresis apparatus and which accommodates a sample gel, and which is adjacent to the gel chamber through a glass filter and which is connected to the gel chamber through a cellophane membrane. a collection chamber that communicates with the opposite electrode of the electrophoresis device and that collects the charged polymer that has passed through the glass filter; and a hollow cell body that accommodates the gel chamber and the collection chamber; The glass filter and cellophane membrane are integrated,
The glass filter is characterized in that it prevents the passage of minute gel fragments but allows the charged polymer to pass through, and the cellophane membrane prevents the charged polymer from passing through.

[For production]

Since the present invention is configured as described above, this collection cell is installed in the electrophoresis tank of an electrophoresis device, and electricity is applied with the gel chamber side as the negative electrode to electrophores the gel containing charged polymers such as DNA. When exposed to water, negatively charged polymers move through the gel to a collection chamber, pass through a glass filter, and are transferred to the collection chamber.

Furthermore, although it tries to move to the positive electrode side, the cellophane membrane in the collection chamber does not allow the charged polymer to pass through the membrane, so the charged polymer remains in the collection chamber. On the other hand, although the microgel fragments also move together with the charged polymer, the glass filter prevents the microgel fragments from entering the collection chamber.

〔Example〕

This invention will be described in more detail with reference to the drawings.

Device Example FIG. 1 is a perspective view showing an example of a collection cell for an electrophoresis device according to the present invention.

The collection cell 13 in this example is composed of a gel chamber 1 that communicates with one electrode of an electrophoresis device and accommodates a sample gel, and a collection chamber 4 adjacent to the gel chamber 1 via a glass filter 2. The chamber 4 communicates with the counter electrode of the electrophoresis device via the cellophane membrane 3, and is for storing and recovering the charged polymer electrophoresed from the gel chamber. The gel chamber 1 and the recovery chamber 4 are housed in a hollow cell main body 5, and a glass filter 2 and a cellophane membrane 3 are integrated into the hollow cell main body 5.

The shape, dimensions, etc. of this collection cell are not limited to this example, and can be changed as appropriate depending on the usage. Similarly, the shape, dimensions, etc. of the gel chamber and the collection chamber can be changed as appropriate depending on the usage. It is desirable to do so.

Various materials can be used for the hollow cell main body as long as they do not contradict the purpose of the collection cell. Examples of such materials include plastics, metals such as stainless steel, glass, and ceramics.

The glass filter used in this invention prevents the passage of minute gel fragments during electrophoresis, and various glass filters can be used in this collection cell as long as they have this function. Similarly, the cellophane membrane prevents the passage of charged polymers during electrophoresis, and various cellophane membranes can be used in this collection cell as long as they have this function.

Example of Use Referring to FIG. 2, an example of using this collection cell will be described.

First, an electrophoresis tank 10 of a horizontal electrophoresis device filled with an electrophoresis buffer 11 is prepared, and a collection cell containing a gel 12 containing a charged polymer of interest in a gel chamber 1 is placed in the electrophoresis tank 10. Set 13. Electrophoresis is performed by applying electricity to the gel chamber 1 side as a negative electrode and the recovery chamber 4 side as a positive electrode. The negatively charged polymer moves in the direction of the collection chamber 4 in the gel, passes through the glass filter 2, and moves to the collection chamber 4. Since the cellophane membrane 3 on the positive electrode side of the recovery chamber does not allow the charged polymer to pass through, the charged polymer remains on the cellophane membrane.

Microgel fragments that move together with charged polymers during electrophoresis cannot pass through the glass filter 2 and remain in the gel chamber 1. After electrophoresis is completed, the charged polymers present on the cellophane membrane are separated from the cellophane membrane by applying current for a short time by reversing the current, and the collected charged polymers are taken out from the collection outlet 14 together with the buffer.

Experimental Example 1 Restriction enzyme 111ndl1 was added to a collection cell as shown in Figure 1.
Approximately 10 μg of the λ-DNA degraded in step 1 was electrophoresed on 1% agarose NA (Pharmacia). Each DN
The agarose block containing the A fragment was cut out, placed in a collection cell as shown in FIG. 1, and subjected to electrophoresis.

The glass filter (GF/F, Whatman) used in this collection cell had the following performance.

The city is 75g/thickness
0.50I1m particle retention capacity (liquid) 0
．． 7μm Light period filtration rate 6.0wl/5ee On the other hand,
Cellophane III (manufactured by Union Carbide, Cellophane Tubing Seamless) had the following performance.

Divided molecular weight 10.000-20.00
0 film thickness 0.020a+■ Using 0.04 mol/g Tris-acetate and 0.001 mol/g EDTA as electrophoresis buffer, 13c
100V in a horizontal electrophoresis device with an electrode distance of m.
For 2 hours, electricity was applied from the outside of the electrophoresis chamber while cooling with ice. After electrophoresis, the DNA was taken out from the collection chamber along with the buffer, treated with phenol and chloroform,
The DNA was precipitated with ethanol and the DNA was re-incubated with 0.
It was dissolved in 1 ml of TE buffer.

To measure the DNA recovery rate, restriction enzyme 111nd
μg of λ-DNA digested with
/10ffl) and 1/10 volume of each of the buffers containing each DNA fragment recovered in the above experiment were subjected to electrophoresis. The results are shown in reference photo 1. Reference photo 1
Lane 1 is a photograph of the gel after electrophoresis placed on a transiminator, and lane 1 shows all DNs before separation.
These are the results of electrophoresis for a sample containing the A fragment, and similarly, lane 2 is 23. IKbp (molecular weight 15.0X
For the DNA fragment of IO6), lane 3 is 9.4 Kb.
Regarding the DNA fragment of p (molecular weight 6.12×10B),
Lane 4 is 6.56 Kbp (molecular weight 4.26 x 106
), lane 5 is 4.36 Kbp.
Lane 6 is for a DNA fragment of 2.32 Kbp (molecular weight 1.51 x 106), and Lane 7 is for a DNA fragment of 2.03 Kbp (molecular weight 1.32 x 106).

The density of the bands in Lane 1 and the corresponding lanes is equal, indicating that all of these DNA fragments were recovered with a 100% recovery rate.

Although the applicable range of DNA separation and recovery described above is from 2381 Kbp to 2.03 Kbp, it can be performed similarly for DNA up to 100 bp.

Experimental Example 2 In order to examine the extent to which enzyme inhibitors were mixed into the DNA collected in this collection cell, 9, 4, which was taken out from the collection chamber,
After purifying the Kbp DNA fragment to various degrees, ligation reaction or restriction enzyme EcoRI cleavage was performed.

The results were measured by agarose electrophoresis. The electrophoresis results are shown in Reference Photo 2. Lane 1 shows the electrophoresis results for the 9.4 Kbp DNA fragment, lane 2 shows the electrophoresis results for the 9.4 Kbp DNA fragment directly ligated, and lane 6 shows the electrophoresis results for the 9.4 Kbp DNA fragment.
The electrophoresis results are shown for a 4 Kbp DNA fragment that was directly digested with the restriction enzyme EcoRI, and lane 4 shows the electrophoresis results for a 9.4 Kbp DNA fragment that was purified by treatment with phenol and chloroform and then subjected to ligage gin. Lane 7 shows the electrophoresis results of a 9.4 Kbp DNA fragment that was once purified by treatment with phenol and chloroform and then digested with the restriction enzyme EcoRI.
The electrophoresis results for NA are shown. This result shows that even a slight amount of gauze or EcoRI inhibitors mixed in the recovered DNA can be easily removed by treatment with phenol or chloroform.

〔Effect of the invention〕

The following effects can be obtained by the collection cell for an electrophoresis apparatus of the present invention.

(1) As demonstrated in the examples, DNA is 100! '
It is possible to recover at a high rate of 6 or close to that.

(2) As demonstrated in the examples, the recovered DNA contains only a trace amount of inhibitor, and this inhibitor can be easily removed, so the recovered DNA can be used for ligation or Genetic manipulations such as restriction enzyme cleavage can be performed.

(3) Since a cellophane membrane is already integrated into the collection cell of the present invention, there is no need for troublesome operations such as supporting a conventional cellophane bag by hand. Furthermore, when taking out the DNA solution, there is no need to open the cellophane bag, and the DNA solution can be taken out simply with a pipettor. As you can see, it has excellent operability.

(4) Since there is no need to touch the cellophane membrane directly with your hands,
It does not contaminate DNA.

(5) The use of radioisotopes (RI) particularly contributes to worker safety.

(6) Treatment of the collection cell contaminated with R1 is simple.

(7) Since the recovery cell of the present invention can be industrially manufactured as a homogeneous product, it enables high recovery rates with good reproducibility.

(8) Since it can be manufactured without particularly requiring expensive materials, an inexpensive collection cell can be obtained.

[Brief explanation of the drawing]

FIG. 1 is an external view showing an embodiment of a collection cell for an electrophoresis device according to the present invention, and FIG. 2 is a sectional view showing electrophoresis using the collection cell according to the invention.

Claims (1)

[Claims] a gel chamber that communicates with one electrode of the electrophoresis device and accommodates a sample gel; and a gel chamber that is adjacent to the gel chamber through a glass filter and communicates with the opposite electrode of the electrophoresis device through a cellophane membrane, and the glass filter It consists of a recovery chamber for recovering the charged polymer that has passed through the gel chamber, and a hollow cell body that accommodates the gel chamber and the recovery chamber, and the cell body, the glass filter, and the cellophane membrane are integrated, and the A collection cell for an electrophoresis device, characterized in that a glass filter prevents the passage of minute gel fragments but allows the charged polymer to pass through, and the cellophane membrane prevents the charged polymer from passing through.