CN114397350B - Preparation method and application of high-concentration agarose electrophoresis gel - Google Patents
Preparation method and application of high-concentration agarose electrophoresis gel Download PDFInfo
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- 229920000936 Agarose Polymers 0.000 title claims abstract description 57
- 238000001962 electrophoresis Methods 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 150000007523 nucleic acids Chemical group 0.000 claims abstract description 23
- 238000000926 separation method Methods 0.000 claims abstract description 17
- 238000001816 cooling Methods 0.000 claims abstract description 4
- 239000000499 gel Substances 0.000 claims description 44
- 239000011543 agarose gel Substances 0.000 claims description 28
- 108020004414 DNA Proteins 0.000 claims description 26
- 108020004707 nucleic acids Proteins 0.000 claims description 16
- 102000039446 nucleic acids Human genes 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 11
- 239000011521 glass Substances 0.000 claims description 9
- 102000053602 DNA Human genes 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 7
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000005266 casting Methods 0.000 claims description 5
- 238000005485 electric heating Methods 0.000 claims description 5
- 238000004090 dissolution Methods 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 6
- 239000012634 fragment Substances 0.000 description 11
- 229920002401 polyacrylamide Polymers 0.000 description 10
- 238000010438 heat treatment Methods 0.000 description 9
- 230000000694 effects Effects 0.000 description 7
- 238000001502 gel electrophoresis Methods 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 238000009835 boiling Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N adenyl group Chemical group N1=CN=C2N=CNC2=C1N GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000007712 rapid solidification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44747—Composition of gel or of carrier mixture
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- Electrochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dispersion Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention belongs to the field of biotechnology, and relates to a preparation method and application of high-concentration agarose electrophoresis gel. The method has the advantages of simple process and convenient operation, the agarose is dissolved in a steam environment under the high-temperature and high-pressure condition to form uniform sol, the uniform sol is poured in the high-temperature environment, and the high-concentration agarose electrophoresis gel is obtained through cooling and solidifying at room temperature, so that the electrophoresis pattern of the obtained high-concentration agarose electrophoresis gel is clear when the electrophoresis separation of the low-molecular-weight nucleic acid fragments is carried out, and the resolution ratio is high.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a preparation method of an electrophoresis separation medium and application thereof, and in particular relates to a preparation method of high-concentration agarose electrophoresis gel and application thereof in low-molecular-weight nucleic acid separation.
Background
Nucleic acids are one of the most basic substances of life, and are classified into ribonucleic acid (abbreviated as RNA) and deoxyribonucleic acid (abbreviated as DNA), wherein DNA is a main substance basis for storing, reproducing and transmitting genetic information, and thus research on DNA is a key point in the field of life science.
Gel electrophoresis technology for separating DNA according to the relative molecular mass size can be used for separating, identifying and purifying DNA fragments (nucleic acid fragments), has been developed as an important experimental means for analyzing and identifying DNA molecules, and is one of core technologies for gene manipulation.
Agarose gel has the advantages of uniform structure, small adsorption to samples, clear electrophoresis pattern, high resolution, good repeatability and the like, and is the most commonly used separation support medium in gel electrophoresis in a laboratory, and is commonly used for separating, identifying and purifying nucleic acid. At present, agarose gel (0.6% -3%) of medium-low concentration is usually prepared in laboratory by heating at normal pressure (heating in a microwave oven), and according to the rule of "the gel concentration is smaller at the time of electrophoresis as the calculated molecular weight is larger", agarose gel of medium-low concentration is often used for separating nucleic acid fragment (DNA fragment) of 100bp-20 kbp. However, in the actual laboratory operation process, the agarose gel with middle and low concentration has poor band separation degree and low resolution for small molecular weight nucleic acid fragments below 200 bp. Thus, high resolution separation of small molecular weight nucleic acid fragments requires higher concentrations (. Gtoreq.4%) of agarose gel. Because of the nature of agarose itself, high concentration agarose gels are difficult to prepare, and laboratory use of polyacrylamide gels instead of high resolution separation of small fractions of nucleic acids has often limited the use of agarose gels in small fractions of nucleic acids, and previous studies have also found that the mobility observed in agarose gels rather than polyacrylamide gels accurately reflects the molecular weight of nucleic acids, and that the presence of consecutive adenine residues in nucleic acid molecules can result in slow movement in polyacrylamide gels, failing to reflect their true size.
Disclosure of Invention
The invention aims to provide a preparation method of high-concentration agarose electrophoresis gel, aiming at the defects of the prior art, which is characterized in that the high-concentration agarose electrophoresis gel is dissolved in a steam environment under high temperature and high pressure conditions to form uniform sol, and the agarose gel is poured into the high-temperature environment to obtain the high-concentration agarose electrophoresis gel, so that the problems that the high-concentration agarose (6 percent or more) is difficult to dissolve, a large number of bubbles are generated, the uniformity is poor and the like when the agarose gel is prepared by a conventional microwave oven heating or boiling heating method are solved.
The invention adopts the technical scheme that:
a preparation method of high-concentration agarose electrophoresis gel comprises the following steps:
1. preparing a buffer solution and an agarose solution: adding 0.1-0.2% NaCl into TBE (Tris-boric acid) as buffer solution for agarose gel preparation; mixing buffer solution and agarose to prepare agarose solution according to the preparation standard of agarose gel with concentration of 6-16%;
2. agarose dissolution: dissolving the prepared agarose solution in a steam environment under high-temperature and high-pressure conditions to obtain agarose sol which is uniformly dissolved;
the high temperature and high pressure conditions in the steam environment are: the temperature is 115-121 ℃ and the pressure is 1.6X10 5 Pa～2.1×10 5 Pa。
3. Agarose electrophoresis gel casting: heating the glass plate glue-pouring device to 75-85 ℃ for preheating, then injecting the agarose sol cooled to 75-85 ℃ into the glass plate glue-pouring device in the environment of 75-85 ℃, inserting a comb, and cooling and solidifying at room temperature to obtain the high-concentration agarose electrophoresis gel.
It is another object of the present invention to provide the use of the high concentration agarose electrophoresis gel described above for the separation of low molecular weight nucleic acids.
The low molecular weight nucleic acid refers to a small molecular weight nucleic acid fragment of 500bp or less.
The low molecular weight nucleic acid refers to single stranded DNA.
The invention has simple process and convenient operation, and can dissolve agarose under high temperature and high pressure condition in steam environment to form uniform sol, thus solving the problems that high concentration agarose (6% or more) is difficult to dissolve and generates a large amount of bubbles and has poor structure uniformity when preparing agarose gel by conventional microwave oven heating or boiling heating method, and simultaneously, the high concentration agarose gel is prepared by casting agarose gel under high temperature environment, thus solving the problems of rapid solidification and poor structure uniformity when casting high concentration agarose gel at room temperature, and the obtained high concentration agarose gel has clear electrophoresis pattern and high resolution when separating low molecular weight nucleic acid fragments by electrophoresis.
Drawings
FIG. 1 shows the dissolution effect of the high concentration agarose sol of the present invention. A: 6g, 8g, 10g, 12g, 14g and 16g agarose are added into 100ml gel buffer; b: and pressurizing and heating to dissolve the sol.
FIG. 2 effects of agarose gel and polyacrylamide gel of different concentrations on the electrophoretic separation of DNA fragments. A: separating DNA fragments by agarose gel; b: isolation of DNA fragments by Polyacrylamide gel. A commercial DNA molecular weight standard of 80-300bp is adopted as Marker, and a 59 bp-204 bp DNA fragment is derived from the amplification of the corresponding length of the ORF region of the HptII gene.
FIG. 3 effect of high concentration agarose gel on single stranded DNA separation. A:10% agarose gel electrophoresis; b:12% agarose gel electrophoresis; c:14% agarose gel electrophoresis; d:14% PAGE gel electrophoresis. The Marker is a commercial DNA molecular weight standard of 80-300bp, and the single-stranded DNA molecules of 19nt to 93nt are synthesized by Shanghai biological engineering company.
Detailed Description
The following describes the embodiments of the present invention in further detail with reference to examples. The following examples are illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer.
1. Preparation of high concentration agarose electrophoresis gel
Examples:
s1, preparing a buffer solution and an agarose solution: 1 XTBE was added with 0.1% NaCl as a buffer for agarose gel preparation; agarose solutions were prepared and placed in reagent bottles (FIG. 1A) based on the gel concentrations of 6%, 8%, 10%, 12%, 14%, and 16% to calculate the amounts of agarose and buffer, respectively.
S2, agarose dissolution: the reagent bottles with the agarose solution are respectively put into an autoclave, and the temperature is set to be 115-121 ℃ (corresponding to the pressure of 1.6x10) 5 Pa～2.1×10 5 Pa), wherein the 6%, 8% agarose solution set temperature is 116 ℃, and the 10%, 12%, 14%, 16% agarose solution set temperature is 121 ℃. Heating for 20min, taking out the reagent bottle when the temperature of the autoclave is reduced to 75-85 ℃, and dissolving agarose to form uniform agarose sol (figure 1B).
S3, agarose electrophoresis gel casting: placing the glass plate glue-pouring device into an electric heating box, preheating at 75-85 ℃, then injecting agarose sol into the glass plate glue-pouring device in the electric heating box (the control temperature is 75-85 ℃), inserting a comb, taking out the glass plate glue-pouring device, and cooling and solidifying at room temperature (about 30 minutes) to obtain high-concentration agarose electrophoresis gels with the concentration of 6%, 8%, 10%, 12%, 14% and 16% (the inner hole specifications of the glass plate glue-pouring devices produced by different manufacturers are different, so the gel specifications are also different).
2. Separating effect of agarose electrophoresis gel with different concentrations on low molecular weight nucleic acid
1. Agarose electrophoresis gels with a length of about 14cm and a concentration of 6%, 8% and 10% respectively were taken, and samples (Marker, DNA fragments of 59bp to 204 bp) were loaded, and electrophoresis was performed at 140V (calculated as gel length of 10V/cm) (the greater the gel concentration, the longer the electrophoresis time required), and after EB staining was performed for 0.5 hours, gel images were taken with OI-100 from Guangzhou optical biosciences Co., ltd (FIG. 2A).
2. Agarose electrophoresis gels of about 14cm in length and 10%, 12% and 14% in concentration were taken, and samples (19 nt to 93nt single-stranded DNA synthesized by Shanghai Biotechnology Co.) were loaded, and electrophoresis was performed at 140V (calculated as gel length of 10V/cm) (the greater the gel concentration, the longer the electrophoresis time required) and then EB stained for 0.5 hour, and gel images were taken with OI-100 from Guangzhou optical Biotechnology Co., ltd. (FIG. 3).
3. Separation effect of polyacrylamide gel with different concentrations on low molecular weight nucleic acid
1. Polyacrylamide electrophoresis gels with a length of about 14cm and a concentration of 10%, 12% and 14%, respectively, were prepared by a conventional method, and samples (DNA fragments of 59bp to 204bp, marker) were loaded, were subjected to electrophoresis at 140V (calculated as gel length of 10V/cm), and were EB-stained for 0.5 hours, and gel images were taken with OI-100 from Guangzhou optical Biotechnology Co., ltd. (FIG. 2B).
Gel electrophoresis separation effect shows that agarose electrophoresis gel and polyacrylamide electrophoresis gel have good separation on the small molecular weight DNA fragments when the commercial DNA molecular weight standard of 80-300 base pairs (bp) is separated (figure 2); the commercial molecular weight standard theoretically has 280, 260, 240 and 220bp 4 bands between 200 and 300bp, the 4 bands are obtained by agarose electrophoresis gel separation (figure 2A), and only 2 bands are obtained by polyacrylamide electrophoresis gel separation (figure 2B); agarose gel background is cleaner (fig. 2); bands of different molecular weight sizes were separated by a greater distance on agarose electrophoresis gels than on polyacrylamide electrophoresis gels (FIG. 2).
The 6% agarose gel can distinguish DNA molecular weight difference of less than 200bp and different from 3bp, the 8% agarose gel can better distinguish molecular weight difference of small fragment DNA of about 100bp (figure 2A), the 10% and above high concentration agarose gel is more suitable for distinguishing nucleic acid difference of smaller molecular weight, and the 10%, 12% and 14% agarose gel has obvious separation effect on single-stranded DNA of 19-93 bases (b) after test (figure 3).
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the scope of the invention.
Claims (1)
1. The use of a high concentration agarose electrophoresis gel in the separation of low molecular weight nucleic acids, characterized in that,
the low molecular weight nucleic acid refers to a small molecular weight nucleic acid fragment below 500bp or a single-stranded DNA of 15-100 nt;
the preparation method of the high-concentration agarose electrophoresis gel comprises the following steps:
1) Preparing a buffer solution and an agarose solution: adding 0.1-0.2% NaCl into TBE as buffer solution for agarose gel preparation; mixing buffer solution and agarose to prepare agarose solution according to the preparation standard of agarose gel with concentration of 6-16%;
2) Agarose dissolution: dissolving the prepared agarose solution in a steam environment under high-temperature and high-pressure conditions to obtain agarose sol which is uniformly dissolved; the high-temperature and high-pressure conditions are as follows: warm temperatureThe temperature is 115-121 ℃ and the pressure is 1.6X10 5 Pa～2.1×10 5 Pa；
3) Agarose electrophoresis gel casting: and (3) placing the glass plate glue-pouring device into an electric heating box, preheating at 75-85 ℃, controlling the temperature of the electric heating box at 75-85 ℃, injecting agarose sol cooled to 75-85 ℃ into the glass plate glue-pouring device in the electric heating box, inserting a comb, taking out the glass plate glue-pouring device, and cooling and solidifying at room temperature to obtain the high-concentration agarose electrophoresis gel.
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Citations (6)
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| CN101173910A (en) * | 2006-10-30 | 2008-05-07 | 姜尚武 | Hemoglobin electrophoresis gelose gel plate and production method thereof |
| CN101701933A (en) * | 2009-10-19 | 2010-05-05 | 王永杰 | non-concentrated urine protein electrophoresis agarose gel plate |
| CN101865877A (en) * | 2010-06-25 | 2010-10-20 | 西北大学 | Gel paving method by using single cell gel electrophoresis |
| CN102532258A (en) * | 2012-03-15 | 2012-07-04 | 中国热带农业科学院热带生物技术研究所 | Method for extracting protein from component with low protein content |
| JP2019028051A (en) * | 2017-07-26 | 2019-02-21 | 国立大学法人広島大学 | Method for producing gel for electrophoresis having high separation ability, and production kit thereof |
| CN110935403A (en) * | 2019-12-07 | 2020-03-31 | 云南省农业科学院生物技术与种质资源研究所 | Method for efficiently preparing agarose gel |
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- 2022-01-14 CN CN202210044239.3A patent/CN114397350B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101173910A (en) * | 2006-10-30 | 2008-05-07 | 姜尚武 | Hemoglobin electrophoresis gelose gel plate and production method thereof |
| CN101701933A (en) * | 2009-10-19 | 2010-05-05 | 王永杰 | non-concentrated urine protein electrophoresis agarose gel plate |
| CN101865877A (en) * | 2010-06-25 | 2010-10-20 | 西北大学 | Gel paving method by using single cell gel electrophoresis |
| CN102532258A (en) * | 2012-03-15 | 2012-07-04 | 中国热带农业科学院热带生物技术研究所 | Method for extracting protein from component with low protein content |
| JP2019028051A (en) * | 2017-07-26 | 2019-02-21 | 国立大学法人広島大学 | Method for producing gel for electrophoresis having high separation ability, and production kit thereof |
| CN110935403A (en) * | 2019-12-07 | 2020-03-31 | 云南省农业科学院生物技术与种质资源研究所 | Method for efficiently preparing agarose gel |
Non-Patent Citations (1)
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