CN108593750B - Method for shortening agarose gel electrophoresis dyeing - Google Patents
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G—PHYSICS
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- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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Abstract
The invention relates to a method for shortening agarose gel electrophoresis dyeing, which belongs to the technical field of biochemical experiments and comprises the steps of installing a gel tray, preparing agarose gel, pouring gel, adding sample, observing and the like.
Description
Technical Field
The invention belongs to the technical field of biochemical experiments, and particularly relates to a method for shortening agarose gel electrophoresis dyeing.
Background
In recent years, molecular biology has been rapidly developed, and Polymerase Chain Reaction (PCR) is a simple and accurate nucleic acid quantitative detection technique. Agarose gel electrophoresis is a standard method for separating, identifying and purifying DNA fragments, and is one of the core techniques of molecular biology. Agarose is a polysaccharide extracted from agar, has hydrophilicity but no electric charge, and is a good electrophoresis support. DNA is negatively charged under alkaline conditions (pH8.0 buffer), moves to the positive pole through a gel medium in an electric field, and different DNA molecular fragments have different migration rates in the electric field due to different molecules and configurations. Ethidium Bromide (EB) can be inserted into base pairs of DNA molecules to form a fluorescent complex, and different zones can be separated after ultraviolet irradiation, so that the purposes of separating, identifying molecular weight, screening and recombining are achieved.
EB is the most commonly used nucleic acid dye in the laboratory, which gives good staining of DNA and RNA in the gel, thus revealing the location of nucleic acids in the gel. But has strong toxicity, strong mutagenicity, moderate carcinogenicity, difficult degradation and transformation and complicated purification treatment, and has great harm to experimenters and the surrounding environment.
Disclosure of Invention
In order to overcome the technical problems in the background technology, the invention provides a method for shortening agarose gel electrophoresis dyeing, which is characterized in that a novel nucleic acid dye GelRed reagent (Biotium) and a 3 × diluted gel sample adding buffer solution are prepared into a mixed solution according to the proportion of 1: 3000-5000 and added into a PCR amplification product, so that the nucleic acid electrophoresis and the dyeing are simultaneously carried out, the process of dyeing the PCR amplification product in the traditional nucleic acid electrophoresis process is cancelled, but the observation is more facilitated.
A method for shortening agarose gel electrophoresis staining, comprising the following steps:
step 1 mounting a gel tray, comprising:
cleaning and airing the measuring cylinder, the triangular flask, the gel tray and the tooth sample grid; sealing two sides of the opening of the gel tray by using adhesive tape paper; placing the gel tray on a horizontal desktop, and inserting a tooth sample grid;
step 2 preparation of the agarose gel, including preparation
Measuring 5 × TBE electrophoresis buffer solution 40m L by using a measuring cylinder, adding water to 200m L, preparing 1 × TBE diluted electrophoresis buffer solution, and pouring the diluted electrophoresis buffer solution into a triangular flask;
weighing 1g-4g of Invitrogen agarose powder, pouring into a triangular flask to prepare agarose gel solution with the concentration of 0.5% -2%, covering kraft paper, and slightly shaking the triangular flask to disperse the agarose powder; putting the triangular flask into a microwave oven, adjusting the gear of the microwave oven to the middle and high fire, and setting the time for 1 min; taking out the triangular flask, slightly shaking the triangular flask, putting the triangular flask into the microwave oven again, adjusting the gear of the microwave oven to the middle-high fire, and setting the time to be 90 s; taking out the triangular flask, slightly shaking the triangular flask until large bubbles are generated, and continuously putting the triangular flask into a microwave oven until agarose is completely dissolved;
step 3, glue filling, comprising
Placing a magnetic stirrer into a triangular flask, placing the triangular flask into a container filled with cold water, stirring and cooling the triangular flask on a magnetic stirrer, slowly pouring the agarose solution into a gel tray when the temperature of the agarose solution in the triangular flask is cooled to 50-60 ℃, so as to avoid generating bubbles, adding a mixed solution of 5 mu L GelRed reagent (Biosum) and 3 × diluted gel loading buffer solution into each 10 mu L DNA sample PCR amplification product, placing the mixture for standby, wherein the 3. mu. 3 × diluted gel loading buffer solution and the GelRed reagent are mixed according to a ratio of 3000-5000: 1, placing the electrophoresis tank on a horizontal table, adjusting the electrophoresis tank to be horizontal, adding 2000m L1 × TBE diluted electrophoresis buffer solution into the electrophoresis tank, tearing sealing adhesive tape after the gel is solidified for 45-60 minutes, transferring the gel tray into the electrophoresis tank, pulling out tooth sample lattices, and cleaning the tooth samples;
step 4, adding the sample, including
Sucking a mixed solution of a PCR amplification product of a 3 mu L DNA sample, GelRed and a 3 × gel sample adding buffer solution, adding the mixed solution into a gel well, adding a DNA molecular weight marker of 3 mu L into a sample well on the left side of a sample to be detected, and using the DNA molecular weight marker as a molecular weight reference;
step 5 electrophoresis, comprising
Covering the cover of the electrophoresis tank, connecting a power supply connecting wire, opening a power supply switch of the electrophoresis apparatus, adjusting the button to an 'on' state and a 'voltage' mode, and selecting a voltage of 100V;
adjusting the voltage gear to 0V, adjusting the button to an off state, and turning off the power switch;
step 6 Observation, including
Opening imaging software in a computer, and connecting a power supply of a gel imaging system; wearing gloves, transferring the gel tray to an ultraviolet lamp table, taking down the gloves, and closing a door of the imaging system; turning on a switch of an ultraviolet lamp, selecting a preview function under a menu of imaging software, adjusting exposure time, clicking a photographing button, acquiring an image and storing the image; closing a switch of the ultraviolet lamp, disconnecting a power supply of the gel imaging system, and opening a door of the imaging system; wearing gloves, taking out the gel from the gel tray, discarding the gel in a garbage bag, wiping the ultraviolet lamp with absorbent paper, and taking down the gloves; the door of the imaging system is closed and the computer is closed.
The further preferred technical scheme is that protective gloves are needed to be worn when the triangular flask is taken out from the microwave oven in the step 2 of preparing the agarose gel.
The further preferable technical scheme is that the stirring speed of the magnetic stirrer in the glue pouring in the step 3 is 300r/min-500r/min, so as to avoid generating bubbles.
The further preferable technical scheme is that 1x TBE dilution buffer solution in the glue pouring in the step 3 needs to be immersed on the surface of the gel by about 1-2 mm.
The invention has the beneficial effects that:
the invention adds the novel nucleic acid dye GelRed reagent into the PCR amplification product, realizes the simultaneous operation of nucleic acid electrophoresis and dyeing, thereby canceling the step of dyeing the PCR amplification product in the traditional nucleic acid electrophoresis process and being more beneficial to observation.
Drawings
FIG. 1 is a diagram of gel electrophoresis using the method of the present invention;
FIG. 2 is a diagram of gel electrophoresis using conventional EB staining;
FIG. 3 is an agarose gel electrophoresis image of nucleic acid detected by a modified EB staining method.
Detailed Description
In order to make the technical scheme, purpose and beneficial effects of the invention clearer, the invention will be described in detail with reference to specific embodiments.
Example 1.
FIG. 1 shows a gel electrophoresis chart of the gel extracted by the method of the present invention.
A method of shortening agarose gel electrophoresis staining comprising: and cleaning the measuring cylinder, the triangular flask, the gel tray and the tooth sample grids, airing, sealing two sides of an opening of the gel tray by using adhesive tape paper, placing the gel tray on a horizontal desktop, inserting the tooth sample grids, and measuring the TBE electrophoresis buffer solution by using the measuring cylinder.
Because the tooth sample grid is flat at one side and concave at the other side, and the gel disk is of a complementary design, the left and right directions need to be noticed when inserting.
Preparation of agarose gel
Weighing 5 × TBE electrophoresis buffer solution 40m L by using a measuring cylinder, adding water to 200m L, preparing 1 × TBE diluted electrophoresis buffer solution, pouring the diluted electrophoresis buffer solution into a triangular flask, weighing 1g to 4g of Invitrogen agarose powder, pouring the Invitrogen agarose powder into the triangular flask to prepare agarose gel solution with the concentration of 0.5 percent to 2 percent, covering kraft paper, and slightly shaking the triangular flask to disperse the agarose powder;
putting the triangular flask into a microwave oven, adjusting the gear of the microwave oven to the middle-high fire (the power is about 80W), setting the time for 1min, taking out the triangular flask by using protective gloves, slightly shaking the triangular flask, putting the triangular flask into the microwave oven again, adjusting the gear to the middle-high fire again, and setting the time for 90 s.
Taking out the triangular flask by using the protective gloves again, and slightly shaking the triangular flask until the powder is completely dissolved when large bubbles are generated; to ensure complete dissolution of the agarose, the agarose must be heated again in the microwave oven until it is completely dissolved.
Putting the magnetic stirrer into a triangular flask, putting the triangular flask into a container filled with cold water, and putting the triangular flask on a magnetic stirrer for stirring and cooling, wherein the stirring speed of the magnetic stirrer is 300r/min-500 r/min. The rotation speed on the magnetic stirrer is not too high so as to avoid generating a lot of bubbles.
Cooling to 50-60 deg.C, slowly pouring the agarose solution into gel tray, placing on hand, and heating without scalding.
Adding a mixed solution of 5 mu L GelRed reagent (Biotium) and 3 × diluted sample adding buffer solution into each 10 mu L DNA sample PCR amplification product, standing for standby use, placing an electrophoresis tank on a horizontal desktop, adjusting the electrophoresis tank to be horizontal, adding 2000m L1 × TBE diluted electrophoresis buffer solution, after about 1 hour, waiting for gel solidification, tearing off adhesive tape paper, transferring a gel tray into the electrophoresis tank, pulling out tooth sample grids, and cleaning, wherein the TBE buffer solution needs to be immersed in the surface of the gel for 1-2 mm.
And sucking a mixed solution of the PCR amplification product of the 3u L DNA sample, the GelRed reagent and the 3 × diluted gel sample adding buffer solution, adding the DNA molecular weight marker of 3u L into a gel pan gel hole on the left side of the sample to be detected, and using the DNA molecular weight marker as a molecular weight reference.
The cover of the electrophoresis tank is covered, a power supply connecting wire is connected, the voltage is selected to be 100V, electrophoresis is started, after electrophoresis is finished, the voltage is firstly reduced to be 0V, then the button is turned to be in a closed state, and finally the power supply switch is turned off.
And (3) opening imaging software in a computer, and connecting a power supply of the gel imaging system. Put on gloves, transfer the gel to the ultraviolet lamp stand, remove the gloves, close the door of the imaging system, at this time can't put on gloves to touch the computer and gel imaging system.
And turning on a switch of the ultraviolet lamp, selecting a preview function under a menu of the imaging software, adjusting exposure time, clicking a photographing button, acquiring an image and storing the image. And closing the switch of the ultraviolet lamp, disconnecting the power supply of the gel imaging system and opening the door of the imaging system. Wearing gloves, taking out the gel, discarding the gel in a garbage bag, wiping the ultraviolet lamp table clean by absorbent paper, taking down the gloves, closing a door of an imaging system, and closing a computer.
Example 2 prior art conventional EB was added directly to the gel
Gel electrophoresis of EB staining is fig. 2.
Preparation of DNA molecular weight standards
Lambda DNA fragments obtained by EcoR I or HindIII enzymes were used as molecular weight standards for electrophoresis. Lambda DNA is a double-stranded DNA molecule of about 50kb in length, and the commercial solution concentration was 0.5mg/m L, and the enzymatic digestion was performed as follows:
HindIII cleaves the DNA to give 8 fragments 23.1, 9.4, 6.6,4.4,2.3,2.0,0.56 and 0.12kb in length.
EcoRI cleaves IDNA to yield 6 fragments of 21.2,7.4,5.8,5.6,4.9 and 2.5kb in length.
Preparation of agarose gel
5 × TBE buffer 20m L was taken and water was added to 200m L to prepare 0.5 × TBE dilution buffer for use.
The preparation of the glue solution comprises weighing 0.4g agarose, placing in 200m L conical flask, adding 0.5 × TBE diluted buffer solution, placing in microwave oven, heating until agarose is completely melted, taking out, shaking to obtain 0.8% agarose gel solution, shaking when not needed during heating to make agarose particles attached to the wall of the flask enter the solution, and covering with sealing film during heating to reduce water evaporation.
Preparing a rubber plate: the two ends of the organic glass groove are respectively sealed by rubber paste. The sealed glue groove is placed on a horizontal support, a sample comb is inserted, and the bottom surface of the glue groove keeps a clearance of about 1mm when observing the lower edge of the sample comb teeth.
Adding Ethidium Bromide (EB) solution into agarose gel liquid cooled to 50-60 ℃ to enable the concentration of the Ethidium Bromide (EB) solution to be 0.5ug/m L, sucking a small amount of melted agarose gel by a pipette to seal the inner side of the rubber paste, carefully pouring residual agarose into a gel tank after the agarose solution is solidified to enable the gel liquid to form a uniform gel layer, wherein the temperature during gel pouring cannot be too low, the solidification is not uniform, the speed is not too high, bubbles are easy to appear, after the agarose gel is completely solidified, the resin is pulled out, the gel at the bottom of a comb is not damaged, and then 0.5 × TBE (tert-butyl ether) is added into the tank to dilute the buffer liquid until the liquid level just does not pass through the upper surface of the gel plate.
And (3) sample adding, namely uniformly mixing 10u L enzymolysis liquid with 2u L6 × sample adding buffer solution, and carefully adding the mixture into a sample groove by using a micro liquid gun, wherein if the content of the DNA is lower, the sample adding amount can be increased according to the proportion, but the total volume cannot exceed the capacity of the sample groove, the gun head needs to be replaced when each sample is added so as to prevent mutual pollution, and the sample adding needs to be carefully operated so as to avoid damaging the gel or puncturing the gel at the bottom of the sample groove.
Electrophoresis: after the sample is added, the electrophoresis tank cover is covered, and the power supply is immediately switched on. The control voltage is kept at 60-80V, and the current is above 40 mA. The electrophoresis was stopped when the bromophenol blue band moved to about 2cm from the gel front.
And (3) dyeing, namely transferring the rubber plate without the EB into EB solution of 0.5ug/m L after the electrophoresis is finished, and dyeing for 20-25min at room temperature.
And (3) observation and photographing: the dyed or EB-loaded electrophoretic gel sheets were observed under a long-wavelength ultraviolet lamp having a wavelength of 254 nm. An orange fluorescent band visible to the naked eye where the DNA was present. When observing under the ultraviolet lamp, protective glasses or an organic glass mask are worn so as to avoid damaging eyes. After the camera lens is added with a close-up lens and a red filter, the camera is fixed on a camera frame, a full-color film is adopted, the aperture is 5.6, and the exposure time is 10-120 seconds.
Preparation of DNA molecular weight standard curve: on the magnified electrophotograph, the migration distance of EcorI and HindIII fragments of lambda DNA in units of centimeters was measured with calipers from the sample well. The common logarithm of the number of nucleotides is used as the ordinate, the migration distance is used as the abscissa, and a smooth curve connecting each point is drawn on a coordinate paper, namely a standard curve of the DNA molecular weight under the electrophoresis condition.
Determination of the size of the DNA fragment: on the amplified electrophoresis photograph, the migration distance of each fragment of the DNA sample is measured by a caliper, and according to the value, the corresponding logarithmic value is checked on the DNA molecular weight standard curve, and the molecular weight of each fragment is further calculated. Conversely, if the size of the DNA fragment is known, the expected migration distance can be determined from the standard curve.
Determination of arrangement sequence of DNA enzyme cutting fragments: and performing logical reasoning according to the electrophoresis analysis results of single enzyme digestion, double enzyme digestion and multiple enzyme digestion by referring to the data of the sizes of the DNA enzyme digestion fragments, and then determining the arrangement sequence of each enzyme digestion fragment and the relative position of each enzyme digestion site. And (4) representing a restriction enzyme map of the DNA molecule by a circular diagram.
Example 3 nucleic acid agarose gel electrophoresis EB staining method improved by the prior art
The modified nucleic acid agarose gel electrophoresis is shown in FIG. 3.
Weighing 1g of agarose, dissolving in 100m L TAE buffer solution, heating in a microwave oven, cooling to about 50 ℃, adding 5u L EB stock solution into the agarose gel to prepare a piece of agarose gel containing 1% of EB, taking 5u L EB stock solution into 95u L distilled water to be marked as EBO, taking 1u L out of the agarose gel, adding the EBO into 1m L bromophenol blue, mixing, marking as EBO1, directly taking a PCR product 5u L and bromophenol blue 3u 84 containing no EB for sample loading, and fully mixing and loading the PCR product 5u L and bromophenol blue containing EB (EBO1)3u L for sample loading, carrying out electrophoresis under the same conditions, carrying out electrophoresis under the voltage of 100V for 15min, and detecting the brightness of the strip under an ultraviolet lamp 35L.
From fig. 1, 2 and 3, it can be seen that the three electrophoretic staining effects are relatively similar, however, the method eliminates the steps of the traditional agarose gel staining process (the gel staining is performed during the electrophoresis process), which not only improves the working efficiency, but also improves the personal safety and the surrounding environment safety of the operators in the electrophoresis experiment.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (3)
1. A method for shortening agarose gel electrophoresis staining is characterized by comprising the following steps:
step 1 mounting a gel tray, comprising:
a. cleaning and airing the measuring cylinder, the triangular flask, the gel tray and the tooth sample grid;
b. sealing two sides of the opening of the gel tray by using adhesive tape paper;
c. placing the gel tray on a horizontal desktop, and inserting a tooth sample grid;
step 2 preparation of agarose gel, comprising
Preparing fresh 5 × TBE electrophoresis buffer solution mother liquor, weighing 54g Tris alkali and 27.5g boric acid, weighing 20M L of prepared 0.5M EDTA solution, and finally diluting to 1000M L with deionized water for later use;
preparing 6 × gel sample-adding buffer solution, namely respectively weighing 25mg of bromophenol blue, 25mg of xylene green FF and 4g of sucrose, and finally fixing the volume to 10m L by using deionized water for later use;
measuring 5 × TBE electrophoresis buffer solution 40m L by using a measuring cylinder, adding water to 200m L, preparing 1 × TBE dilution buffer solution and pouring the buffer solution into a triangular flask;
weighing 1g-4g of Invitrogen agarose powder, pouring into a triangular flask to prepare agarose gel solution with the concentration of 0.5% -2%, covering kraft paper, and slightly shaking the triangular flask to disperse the agarose powder;
putting the triangular flask into a microwave oven, adjusting the gear of the microwave oven to the middle and high fire, and setting the time for 1 min;
taking out the triangular flask, slightly shaking the triangular flask, putting the triangular flask into the microwave oven again, adjusting the gear of the microwave oven to the middle-high fire, and setting the time to be 90 s;
taking out the triangular flask, slightly shaking the triangular flask until large bubbles are generated, and continuously putting the triangular flask into a microwave oven until agarose is completely dissolved;
step 3, glue filling, comprising
a. Putting the magnetic stirrer into a triangular flask, putting the triangular flask into a container filled with cold water, putting the triangular flask on a magnetic stirrer, stirring and cooling,
the stirring speed of the magnetic stirrer is 300r/min-500r/min to avoid generating bubbles;
b. when the temperature of the agarose solution in the triangular flask is cooled to 50-60 ℃, slowly pouring the agarose solution into a gel tray so as to avoid generating bubbles;
c. adding a mixed solution of 5 mu L GelRed reagent and 3 × diluted gel loading buffer solution into each 10 mu L DNA sample PCR, and placing for later use, wherein the 3 × diluted gel loading buffer solution and the GelRed staining solution are mixed according to a mixing ratio of 3000-5000: 1;
d. placing the electrophoresis tank on a horizontal desktop, adjusting the electrophoresis tank to be in a horizontal state, and adding 1 × TBE (tunnel boring machine) of 2000m L to dilute the electrophoresis buffer solution into the electrophoresis tank;
e.45-60 minutes after the gel is solidified, tearing the adhesive tape sealing paper, transferring the gel tray into an electrophoresis tank, pulling out the tooth sample grids, and cleaning the tooth sample grids;
step 4, adding the sample, including
Sucking a mixed solution of a PCR amplification product of a 3 mu L DNA sample, a GelRed reagent and a 3 × diluted gel sample adding buffer solution, adding the mixed solution into an agarose gel well, adding a DNA molecular weight marker of 3 mu L into a sample well on the left side of a sample to be detected, and using the DNA molecular weight marker as a molecular weight reference;
step 5 electrophoresis, comprising
Covering the cover of the electrophoresis tank, connecting a power supply connecting wire, opening a power supply switch of the electrophoresis apparatus, adjusting the button to an 'on' state and a 'voltage' mode, and selecting a voltage of 100V;
adjusting the voltage gear to 0V, adjusting the button to an off state, and turning off the power switch;
step 6 Observation, including
a. Opening imaging software in a computer, and connecting a power supply of a gel imaging system;
b. wearing gloves, transferring the gel tray to an ultraviolet lamp table, taking down the gloves, and closing a door of the imaging system;
c. turning on a switch of an ultraviolet lamp, selecting a preview function under a menu of imaging software, adjusting exposure time, clicking a photographing button, acquiring an image and storing the image;
d. closing a switch of the ultraviolet lamp, disconnecting a power supply of the gel imaging system, and opening a door of the imaging system;
e. wearing gloves, taking out the gel from the gel tray, discarding the gel in a garbage bag, wiping the ultraviolet lamp with absorbent paper, and taking down the gloves;
f. the door of the imaging system is closed and the computer is closed.
2. The method for shortening agarose gel electrophoresis dyeing according to claim 1, wherein the agarose gel preparation of step 2 is carried out by taking out the triangular flask from the microwave oven while wearing protective gloves.
3. The method for shortening agarose gel electrophoresis staining of claim 1, wherein the 1x TBE dilution buffer solution in the gel filling of the step 3 needs to be immersed on the surface of the gel for 1-2 mm.
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| CN106191244A (en) * | 2016-07-11 | 2016-12-07 | 天津金域医学检验所有限公司 | A kind of method of DNMT3A gene test |
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