CN-116286790-B - DNA (deoxyribonucleic acid) purifying kit and DNA purifying method by using ultra-trace agarose gel

CN116286790BCN 116286790 BCN116286790 BCN 116286790BCN-116286790-B

Abstract

The invention provides an ultra-trace agarose gel purification DNA kit and a DNA purification method, and relates to the technical field of biology, wherein the kit comprises sol solution, impurity removal solution, rinsing solution, elution buffer solution, an ion adsorption column and a collection pipe; the impurity removing liquid comprises 4-6M urea, 2-3M NaCl and 70-80% absolute ethyl alcohol, the ion adsorption column adopts an upper layer nano film and a lower layer nano film, the upper layer nano film is a polycarbonate film, the lower layer nano film is a polyamide film, and the kit is low in cost and high in recovery efficiency. The invention also discloses a method for purifying DNA, which comprises the steps of sol, impurity removal, rinsing, elution and the like through a unique buffer solution system and a centrifugal adsorption column method, is used for recovering agarose gel DNA and PCR products, and has the advantages of simple and quick operation, high recovery efficiency, safety and environmental protection.

Inventors

LI YANYAN

Assignees

核小体(北京)生物技术有限公司

Dates

Publication Date: 20260505
Application Date: 20230112

Claims (4)

1. The DNA kit for ultra-trace agarose gel purification is characterized by comprising a sol solution, a impurity removing solution, a rinsing solution, an elution buffer solution, an ion adsorption column and a collecting pipe, wherein the concentration of the impurity removing solution is 5-5.5M urea, 2-3M NaCl and 70-80% absolute ethyl alcohol, the ion adsorption column adopts an upper layer nano film and a lower layer nano film of the ion adsorption column, the upper layer nano film of the ion adsorption column is a polycarbonate film, the lower layer nano film is a polyamide film, and the micropore diameter of the polycarbonate film is less than 5nm; wherein, 2.2M NaCl and 70% absolute ethyl alcohol; Wherein the sol solution comprises 50-100mM Tris-HCl buffer solution and 7% phenol red solution, and the pH of the Tris-HCl buffer solution is=8.0; wherein the rinsing liquid comprises 70% -80% absolute ethyl alcohol, and the pH value is 7.5; wherein the elution buffer comprises 1mM-3mM Tris-HCl, pH=8.0-9.0.
2. The ultra-trace agarose gel purification DNA kit according to claim 1, wherein the 7% phenol red solution is prepared by weighing 0.1 g of phenol red, placing in a mortar, adding 5.7 ml of 50mM NaOH solution and a proper amount of water, grinding the phenol red, and keeping the volume to 250ml and 4 ℃.
3. A method for purifying DNA using the ultra-minimal quantity agarose gel purification DNA kit of claim 1, comprising the steps of: cutting the target DNA band from agarose gel under the long-wave ultraviolet lamp; adding sol solution, namely placing the target DNA strip into a 1.5 ml centrifuge tube, and adding 400-500 mu L of sol solution; Sol, namely placing the centrifuge tube in a water bath environment with the temperature of 50-65 ℃ until the target DNA strip is completely dissolved, and uniformly mixing every 2-3min during the water bath; Adjusting sol solution, namely titrating NaOH solution to adjust the pH of the sol solution, and stopping titrating when the sol solution turns into mauve; adding 500 mu L of impurity removing liquid into the mixed liquid, uniformly mixing, and centrifuging at 12,000rpm for 1min to obtain a supernatant solution; loading the supernatant into ion adsorption column in collecting pipe, standing at room temperature for 1min, centrifuging at 12,000rpm for 30s, and discarding the waste liquid; rinsing, namely adding 600 mu L of rinsing liquid into the ion adsorption column, centrifuging at 12,000rpm for 30s, discarding waste liquid, and repeating rinsing once; Removing the rinse liquid, namely centrifuging the ion adsorption column at 12,000rpm for 2min, and discarding the waste liquid; Eluting, namely taking out the ion adsorption column from the collecting pipe, putting the ion adsorption column into a new centrifuge tube, adding 50 mu L of eluent into the middle part of the ion adsorption column, standing for 2min at room temperature, and centrifuging for 1min at 12,000 rpm.
4. A method of purifying DNA according to claim 3, wherein the agarose gel is a PCR product, comprising the steps of: cutting gel, namely cutting a target PCR band from agarose gel under a long-wave ultraviolet lamp; adding sol solution, namely placing the target PCR strip into a 1.5 ml centrifuge tube, supplementing the PCR reaction solution or the enzyme digestion reaction solution to 100 mu L by using water, adding 300 mu L of sol solution, and uniformly mixing; Adjusting sol solution, namely titrating NaOH solution to adjust the pH of the sol solution, and stopping titrating when the sol solution turns into mauve; Adding 500 μl of impurity removing liquid into the mixed solution, mixing, centrifuging at 12,000rpm for 1min to obtain supernatant, loading into ion adsorption column in collecting tube, standing at room temperature for 1min, centrifuging at 12,000rpm for 30s, and discarding the waste liquid; rinsing, namely adding 600 mu L of rinsing liquid into the ion adsorption column, centrifuging at 12,000rpm for 30s, discarding waste liquid, and repeating rinsing once; Removing the rinse liquid, namely centrifuging the ion adsorption column at 12,000rpm for 2min, and discarding the waste liquid; Eluting, namely taking out the ion adsorption column from the collecting pipe, putting the ion adsorption column into a new centrifuge tube, adding 50 mu L of eluent into the middle part of the ion adsorption column, standing for 2min at room temperature, and centrifuging for 1min at 12,000 rpm.

Description

DNA (deoxyribonucleic acid) purifying kit and DNA purifying method by using ultra-trace agarose gel Technical Field The invention relates to the technical field of biology, in particular to an ultra-trace agarose gel purified DNA kit and a DNA purification method. Background Purification and analysis of DNA are widely used in the field of molecular biology, and in the genetic cloning technology, the amplified products of the polymerization chain reaction are usually subjected to analysis such as recovery, purification and sequencing, or the linearized plasmid DNA is recovered and purified by digestion of circular plasmids for subsequent molecular experiments. Common methods for recovering DNA fragments from agarose gel include DEAE-cellulose filter recovery, low melting point agarose, electric dialysis bag washing, hole digging, etc. However, the methods have the problems of complicated experimental operation, low DNA recovery efficiency, high cost and the like, and the column method for recovering DNA is still the most widely applied method with high operation efficiency. However, the column method has disadvantages such as low recovery amount, more adsorption columns are needed if a large amount of nucleic acid is required to be recovered, and the existing adsorption columns are mainly made of silica hydroxyl groups, which is a main cause of high cost for recovering DNA by the column method. Disclosure of Invention The invention aims to provide an ultra-trace agarose gel purified DNA kit and a method for purifying DNA, so as to solve the problems. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: The application provides an ultra-trace agarose gel purification DNA kit which is characterized by comprising a sol solution, a impurity removing solution, a rinsing solution, an elution buffer solution, an ion adsorption column and a collecting pipe, wherein the impurity removing solution comprises 4-6M urea, 2-3M NaCl and 70-80% absolute ethyl alcohol, the ion adsorption column adopts an upper layer nano film and a lower layer nano film, and the nano film has certain flexibility and rigidity, has a plurality of ionic groups, and is resistant to strong acid, strong alkali, high salt and ethanol solution. Further, the upper layer of the nanometer membrane of the ion adsorption column is a polycarbonate membrane, the diameter of micropores of the polycarbonate membrane is smaller than 5nm, urea and NaCl are filtered, the lower layer of the nanometer membrane is a polyamide membrane, the polyamide membrane contains hydrophilic amide groups, and can form hydrogen bonds with DNA to combine, so that the effect of intercepting the DNA is achieved, the stronger the hydrogen bond capability is, the stronger the DNA combining capability is, and the purpose of separating salt solution and DNA is achieved. And finally, eluting the pure DNA from the nano membrane under the condition of pH higher than that of the rinsing liquid. Further, the concentration of the impurity removing liquid is 5-5.5M urea, 2.2M NaCl and 70% absolute ethyl alcohol. The urea can enable nonspecific proteins on DNA to be in a denatured state, and can well protect the structural stability of nucleic acid. The NaCl solution dissolves DNA and is separated from protein by centrifugation. The salt component in the aqueous phase of DNA was washed with 70% absolute ethanol. Further, the sol solution comprises 50-100mM Tris-HCl buffer solution and 7% phenol red solution, wherein the pH of the Tris-HCl buffer solution is=8.0, and the Tris-HCl buffer solution is used for protecting the stability of DNA. Further, the preparation method of the 7% phenol red solution comprises the steps of weighing 0.1g of phenol red, placing the phenol red into a mortar, adding 5.7ml of 50mM NaOH solution and a proper amount of water, grinding the phenol red, and preserving the phenol red at a constant volume of 250ml and at a temperature of 4 ℃. The phenol red solution is used as an acid-base indicator, and is yellow when the pH value is lower than 6.4, and the acid-base indicating characteristic of the phenol red solution is utilized to judge whether the sol solution is deteriorated or not, observe the sol effect and monitor the pH value change, so that the optimal combination effect is achieved, and the recovery efficiency is improved. Further, the rinse solution comprises 70% -80% absolute ethanol, and the pH=7.5. The 70% -80% absolute ethyl alcohol can effectively remove salt solution and other impurities and precipitate DNA. Further, the elution buffer comprises 1mM-3mM Tris-HCl, pH=8.0-9.0, for eluting pure DNA from the nanomembrane, preferably at a concentration of 1mM. A method of purifying DNA comprising the steps of: Cutting gel, namely cutting the target DNA strip from agarose gel under a long-wave ultraviolet lamp, and cutting the gel without DNA as far as possible; Adding sol solution, namely placing the target DNA