CN108795925A - DNA glue extraction agent and the method for using the reagent purification PCR product - Google Patents

Abstract

The invention discloses a DNA gel extraction reagent and a method for purifying PCR products using the reagent. It comprises agarose gel dissolving liquid and washing liquid for DNA gel extraction, and the formula of the DNA gel extracting agarose gel dissolving liquid is as follows: : 6mol/L guanidine hydrochloride, 25mmol/L sodium citrate, 0.5% lauroyl sarcosine, 0.1mol/L 2-mercaptoethanol; the formula of the washing liquid is: 10mmol/L Tris.HCl, 100mmol/L NaCl, 1mmol /L EDTA, volume concentration 80% ethanol, the pH value of Tris.HCl is 8.0. The DNA gel extraction reagent of the present invention is used together with the regenerated silicon column, which can reduce the number of disposable commercial kits used, reduce the generation of laboratory waste, protect the environment, and save social resources and use costs.

Description

DNA gel extraction reagent and method for purifying PCR product using the reagent

technical field

The invention belongs to the field of biological reagents, in particular to a DNA gel extraction reagent and a method for purifying PCR products using the reagent.

Background technique

Gene cloning (also known as molecular cloning) is the most commonly used technique in modern molecular biology, widely used in biology, medicine, biopharmaceutical industry and other fields. Gene cloning refers to the process of recombining the obtained target gene or DNA (deoxyribonucleic acid) fragment with the plasmid (vector) in vitro and introducing the recombinant vector into the host for replication, and obtaining a large amount of recombinant DNA by extracting the plasmid. This process includes amplification and purification of the target gene, restriction enzyme digestion reaction, agarose electrophoresis and DNA gel extraction or purification of the digested product, ligation of the target DNA with the plasmid, bacterial transformation of the ligated product, bacterial culture and recombinant plasmid extraction and other major steps. Among them, DNA purification plays a key role in the efficiency of gene cloning. In the process of gene cloning, DNA gel extraction kits are often used to extract and purify DNA from agarose after electrophoresis of digested DNA. Using the DNA gel extraction kit to purify DNA can not only improve the quality of DNA, but also greatly accelerate the efficiency of gene cloning and the process of research. At present, DNA gel extraction kits are commonly used in most laboratories around the world, and they are used in large quantities, but they are also one of the main expenditures of laboratories. Although DNA gel extraction kits have produced huge economic benefits in the market, since these kits are mainly designed and prepared for one-time use, the use of the kits not only produces a large amount of laboratory waste, but also causes a waste of resources. Therefore, the purpose of the present invention is to reduce the consumption of the DNA gel extraction kit, protect the environment, and save money for the laboratory.

Contents of the invention

The technical problem to be solved by the present invention is: how to provide a DNA gel extraction reagent to solve the problems of environmental pollution and waste caused by the one-time use of the existing DNA gel extraction kits.

The technical scheme of the present invention is: agarose gel solution for DNA gel extraction, its formula is: 6mol/L guanidine hydrochloride, 25mmol/L sodium citrate, 0.5% lauroyl sarcosine, 0.1mol/L 2- Mercaptoethanol.

DNA gel extraction reagent, it comprises DNA gel extraction with agarose gel solution and washing solution, the formula of DNA gel extraction with agarose gel solution is: 6mol/L guanidine hydrochloride, 25mmol/L sodium citrate, 0.5 %lauroyl sarcosine, 0.1mol/L 2-mercaptoethanol; the formula of the washing liquid is: 10mmol/L Tris.HCl, 100mmol/L NaCl, 1mmol/L EDTA, 80% ethanol by volume concentration, the pH value of Tris.HCl is 8.0.

The invention also discloses a method for gel extraction and purification of PCR products, comprising the following steps:

(1) Carry out agarose gel electrophoresis to the PCR amplification product;

(2) After the electrophoresis is completed, cut out the gel block containing DNA, weigh the weight of the cut gel block, and place the gel block in a sterile centrifuge tube;

(3) In the centrifuge tube, add the above-mentioned agarose gel solution for DNA gel extraction of three times the volume of the gel block weight, and heat the gel in a 75°C metal bath to dissolve the gel; wherein the gel block weight is in milligrams, The volume of agarose gel solution for DNA gel extraction is in microliters;

(4) After the glue is completely dissolved, cool to room temperature, add isopropanol of 1 times the weight of the glue to the centrifuge tube and mix; the weight of the glue block is in milligrams, and the volume of isopropanol is in microliters;

(5) Transfer the gel mixture in the centrifuge tube to a silicon column with a collection tube, place it in a centrifuge, centrifuge at 12000rpm for 1 minute, and discard the waste liquid in the collection tube;

(6) Add the above-mentioned washing solution to the silicon column, centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, put the silicon column back into the collection tube, without adding any liquid, and centrifuge again at the same speed for 1 minute to finish Remove the residual liquid in the silicon column;

(7) Place the silicon column in a sterile centrifuge tube, add deionized distilled water to the silicon column, and after 1 minute at room temperature, centrifuge at 12000 rpm for 1 minute;

(8) The DNA sample collected in the centrifuge tube was tested.

Further, in order to achieve the purpose of material saving in the present invention, the silicon column is a regenerated silicon column.

Further, the regenerated silicon column is prepared by the method of claim 2 in the patent application number: 201710437190.7. which is:

(1)) Take the used disposable silicon column and put it in the collection tube, add 0.75mL distilled water, carry out centrifugation and discard the waste liquid;

(2) Add 0.5mL of 1mol/L phosphoric acid solution, let it stand for 3 minutes for centrifugation and discard the waste liquid; repeat this step five times;

(3) Wash the silicon column with 0.75mL distilled water, perform centrifugation and discard the waste liquid;

(4) Move the silicon column to a sterile centrifuge tube, and centrifuge again to remove all the residual moisture in the silicon column to obtain a regenerated silicon column.

Compared with the prior art, the present invention has the following beneficial effects:

The DNA gel extraction reagent of the present invention can be successfully used for DNA purification without affecting the yield of DNA purification. The DNA gel extraction reagent of the present invention is used together with the regenerated silicon column, which can reduce the number of disposable commercial kits used, reduce the generation of laboratory waste, protect the environment, save social resources and use costs.

Description of drawings

Fig. 1 is the comparison of the DNA gel extraction effect of the regenerated silicon column combined with the DNA gel extraction reagent of the present invention and the original silicon column and reagent of the kit on the DNA gel extraction effect.

Detailed ways

Embodiment 1DNA gel extraction uses the configuration of agarose gel solution

The agarose gel solution includes 6 mol/L Guanidine.HCl, 25 mmol/L Sodium Citrate, 0.5% N-Lauroylsarcosine, and 0.1 mol/L 2-mercaptoethanol.

Taking 100 ml of agarose gel solution as an example, the preparation method is as follows:

(1) Weigh 57.3 grams of guanidine hydrochloride, 7.35 grams of sodium citrate and 0.5 grams of lauroyl sarcosine respectively in an electronic balance, place the medicine in a beaker with a capacity of 250 milliliters, add 50 milliliters of distilled water, and place on a magnetic stirrer Heat to dissolve.

(2) Take 6.8 microliters of 2-mercaptoethanol with a 20 microliter pipette gun, add it to the above solution, stir and mix well.

(3) Transfer the solution to a 100ml graduated cylinder, add distilled water to make it 100ml. Divide the volume-contained solution into 50 ml sterile centrifuge tubes, and seal them for storage.

The configuration of embodiment 2 washing liquid

The washing liquid contains 10mmol/L Tris.HCl (pH 8.0), 100mmol/L NaCl, 1mmol/L EDTA, 80% ethanol.

Take 100ml washing liquid as an example below, the preparation method is as follows:

(1) Prepare 1mol/L Tris.HCl (pH 8.0) mother liquor and 0.5mol/L EDTA mother liquor according to conventional methods;

(2) Weigh 0.585 gram of NaCl in electronic balance, place in 250 milliliter reagent bottle;

(3) Add 1 ml of 1mol/L Tris.HCl (pH8.0) mother solution and 200 microliters of 0.5mol/LEDTA mother solution to the reagent bottle;

(4) Add 18.5 milliliters of distilled water to completely dissolve NaCl in the reagent bottle;

(5) Measure 80 ml of absolute ethanol with a graduated cylinder, add it to the reagent bottle, mix thoroughly with the solution, and seal it with a cap.

Experimental example

In order to identify the effect of the DNA gel extraction reagent of the present invention on DNA gel extraction or purification, utilize the regenerated silicon column in the Qiagen and Axygen gel extraction kits (the preparation method of the regenerated silicon column refers to the patent application number: 201710437190.7, and the name of the invention is : silicon column rapid regeneration method) and reagent of the present invention are purified to the DNA gel after electrophoresis; Simultaneously compare with the DNA purification effect of Qiagen and Axygen DNA gel extraction kit original silicon column and reagent.

Utilize reagent of the present invention to complete DNA gel extraction method as follows:

(1) Prepare 1% agarose gel according to the usual method. After the gel is cooled, pull out the loading comb and place it in the electrophoresis tank containing 1xTAE, load 50 microliters of PCR product (LIF DNA) in the loading hole, Perform electrophoresis at 100V for 20 minutes;

(2) After the electrophoresis is over, the DNA-containing gel block is cut out under an ultraviolet lamp, the weight of the cut gel block is weighed in an electronic balance, and the gel block is placed in a 1.5 ml sterile centrifuge tube;

(3) Add the agarose gel solution (calculated according to the ratio of 100 milligrams of glue plus 100 microliters of gel solution) for DNA gel extraction of the present invention in centrifuge tubes, in a 75°C metal bath Heat to dissolve the glue;

(4) After the glue is completely dissolved, cool to room temperature, add isopropanol of 1 times the weight of the glue to the centrifuge tube and mix.

(5) Transfer the gel mixture in the centrifuge tube to the regenerated silicon column (note: the regenerated silicon column comes with a collection tube), place it in a centrifuge, centrifuge at 12000rpm for 1 minute, and discard the waste liquid in the collection tube;

(6) Add 600 ml of washing liquid of the present invention to the regenerated silicon column, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid in the collection tube. Put the regenerated silicon column back into the collection tube without adding any liquid, and centrifuge again at the same speed for 1 minute to remove the residual liquid in the regenerated silicon column;

(7) Place the regenerated silicon column in a 1.5 ml sterile centrifuge tube, add 35 microliters of deionized distilled water to the regenerated silicon column, and after 1 minute at room temperature, centrifuge at 12000 rpm for 1 minute;

(8) The DNA sample collected in the centrifuge tube was tested.

Qiagen and Axygen DNA Gel Extraction Kit original silica column and reagent purification methods are completed according to the respective experimental manuals. The obtained samples were quantified and detected by electrophoresis.

The result shows that the regenerated silicon column cooperates with the DNA gel extraction reagent of the present invention to the DNA gel extraction effect and the extraction effect of the original silicon column of the kit and the reagent and has no significant difference (Fig. 1); the DNA gel extraction reagent of the present invention is illustrated Using it with the regenerated silica column does not affect the yield of DNA purification, which proves that the agarose gel solution used for DNA gel extraction can extract DNA from the agarose gel and has a good DNA extraction effect.

The above-mentioned embodiments only express the specific implementation manners of the present application, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the protection scope of the present application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the technical solution of the present application, and these all belong to the protection scope of the present application.

Claims (5)

1.DNA glue extracting agarose gel lysate, which is characterized in that its formula is：6mol/L guanidine hydrochlorides, 25mmol/L lemons Lemon acid sodium, 0.5% Hamposyl L, 0.1mol/L 2 mercapto ethanols.

2.DNA glue extraction agents, which is characterized in that it includes DNA glue extracting agarose gel lysate described in claim 1 And cleaning solution, the formula of cleaning solution are：10mmol/L Tris.HCl, 100mmol/L NaCl, 1mmol/L EDTA, volume are dense 80% ethyl alcohol is spent, the pH value of Tris.HCl is 8.0.

3. a kind of method of PCR product glue extracting and purifying, which is characterized in that include the following steps：

(1) pcr amplification product is subjected to agarose gel electrophoresis；

(2) electrophoresis terminates, and the blob of viscose containing DNA is cut out, and weighs the weight for cutting out blob of viscose, blob of viscose is placed in sterile centrifugation tube；

(3) right that the three times volume of blob of viscose weight is added into centrifuge tube wants the DNA glue extractings described in 1 to be dissolved with agarose gel Liquid heats in 75 DEG C of metal baths by peptization solution；Wherein blob of viscose weight is in terms of milligram, DNA glue extracting agarose gel lysate Volume by microlitre in terms of；

(4) it after glue is completely dissolved, is cooled to room temperature, into centrifuge tube plus the isopropanol of 1 times of volume of glue weight mixes；Wherein Blob of viscose weight in terms of milligram, isopropanol volume by microlitre in terms of；

(5) the glue mixed liquor in centrifuge tube is moved in the silicon column of subsidiary collecting pipe, is placed in a centrifuge, with 12000rpm speed Centrifugation 1 minute, abandons waste liquid in collecting pipe；

(6) cleaning solution into silicon column plus described in claim 2 is centrifuged 1 minute with 12000rpm speed, is abandoned in collecting pipe and is given up Silicon column is put back to collecting pipe by liquid, is not added with any liquid, and 1 minute is centrifuged again to eliminate Liquid Residue in silicon column with identical speed；

(7) silicon column is placed in sterile centrifugation tube, into silicon column plus deionized-distilled water, room temperature are fast with 12000rpm after 1 minute Degree centrifugation 1 minute；

(8) DNA sample collected in centrifuge tube is detected.

4. a kind of method of PCR product glue extracting and purifying according to claim 3, which is characterized in that the silicon column is Regenerate silicon column.

5. the method for a kind of PCR product glue extracting and purifying according to claim 4, which is characterized in that the regeneration silicon Column is prepared by the following method：

(1) it takes the disposable silicon column after use to be placed in collecting pipe, adds 0.75mL distilled water, be centrifuged and discard useless Liquid；

(2) 1mol/L phosphoric acid solution 0.5mL are added, stands 3 minutes and is centrifuged and discards waste liquid；Repeat the step 5 It is secondary；

(3) silicon column is cleaned with 0.75mL distilled water, be centrifuged and discard waste liquid；

(4) silicon column is moved in sterile centrifugation tube, is centrifuged again, eliminate in silicon column residual moisture to get to regeneration silicon column.