Method and technique for detecting DNA by agarose gel electrophoresis
[Objective requirements]
Method and technique for detecting DNA by agarose gel electrophoresis through this experiment
[Experimental principle]
Agarose gel electrophoresis is a common method for isolating and identifying DNA fragments. The DNA molecule has a charge effect and a molecular sieve effect when it moves in an agarose gel. The DNA molecule is negatively charged in a pH solution higher than the isoelectric point and moves toward the positive electrode in the electric field. Due to the structurally repetitive nature of the sugar phosphate backbone, the same amount of double-stranded DNA has almost the same amount of net charge, so they can move toward the positive electrode at the same rate. Different concentrations of agarose gel can separate DNA fragments from 200 bp to 50 kb. A low concentration of ethidium bromide (EB) was added to the agarose solution, and 10 ng of DNA bands were detected under ultraviolet light. In the electric field, pH 8.0, the gel was negatively charged. DNA migrates to the anode.
Agarose gel has the following characteristics:
(1) Molecular size of DNA The rate of migration in the gel matrix is ​​inversely proportional to the usual logarithm of the number of base pairs, and the larger the molecule, the slower the migration.
(2) Agarose concentration A linear DNA molecule of a specific size whose migration speed is different in different concentrations of agarose gel. The logarithm of the DNA electrophoretic mobility (u) is linear with the gel concentration (t).
(3) When the voltage is low, the linear DNA fragment migration rate is proportional to the applied voltage. However, as the electric field strength increases, the mobility of DNA fragments of different molecular weights will increase with different amplitudes. With the increase of voltage, the effective separation range of agarose gel will shrink. To maximize the resolution of DNA fragments larger than 2 kb, the applied voltage must not exceed 5 v/cm.
(4) Electrophoresis temperature The electrophoretic behavior of DNA in agarose gel electrophoresis is not affected by the temperature during electrophoresis. The relative migration rate of DNA fragments of different sizes does not change significantly between 4 °C and 30 °C, but the concentration is low. The 0.5% gel or low melting gel is more fragile and is preferably electrophoresed at 4 °C.
(5) Embedding dye fluorescent dye Ethidium bromide is used to detect DNA in agarose gel. The dye is embedded in the stacked base pairs and stretches the linear and notched circular DNA to make it more rigid. Will reduce the linear mobility by 15%.
(6) The composition of the ionic strength electrophoresis buffer and its ionic strength affect the mobility of DNA electrophoresis. In the absence of ions (such as misuse of distilled water to prepare the gel, the conductivity is the smallest, the DNA is almost not moving, in the high ionic strength buffer (such as adding 10 × electrophoresis buffer), the conductance is very high and the heat is obviously In severe cases, the gel will melt.
For the natural double-strand, several commonly used electrophoresis buffers are TAE, TBE, etc., which are generally formulated into a concentrated mother liquor, stored at room temperature, and diluted at the time of use.
[Experimental Instruments and Equipment]
1. Constant temperature incubator 2. Agarose gel electrophoresis system
3. Autoclave 4. UV Transmitter
[Experimental Materials]
1. Tris) 2. Boric acid
3. Ethylenediaminetetraacetic acid (EDTA) 4. Bromophenol blue
5. Sucrose 6. Agarose
7. Ethidium bromide 8.DNA marker
9.DNA sample
[Experimental steps]
1. Preparation of buffer
5 x TBE (5 volumes of TBE stock solution)
With 1000ml 5×TBE:
Tris 54g
Boric acid 27.5g
0.5mol/l EDTA 20ml
Ph8.0
Gel Loading Buffer (6×)
Bromophenol blue 0.25%
Sucrose 40%
3 ethidium bromide solution (EB) 0.5μg/ml
2. Preparation of agarose gel
The percentage of agarose in the gel is determined according to the size of the DNA to be separated. Can refer to the following table:
Effective separation range of agarose gel concentration linear DNA
0.3% 5-60 kb
0.6% 1-20 kb
0.7% 0.8-10 kb
0.9% 0.5-7 kb
1.2% 0.4-6 kb
1.5% 0.2-4 kb
2.0% 0.1-3 kb
3. Preparation of rubber sheet
(1) Seal the edge of the washed and dried glass plate (or the opening of the plastic plate prepared by the electrophoresis device) with an autoclave indicating tape to form a film (put the film on the level of the table) Position, correct with the level).
(2) Prepare a running buffer (1 × TBE) sufficient for filling the electrophoresis tank and preparing the gel. Accurately weigh the agarose powder. The buffer should not exceed 50% of the volume of the cone or glass bottle. It is important to use the same batch of running buffer in the electrophoresis tank and gel. Small differences in ionic strength or pH will form a leading edge in the gel, which will greatly affect the mobility of DNA fragments.
(3) Loosely wrap a thick layer of paper on the bottle neck of the cone. If a glass bottle is used, the cap must be loosened. The suspension is heated to agarose dissolution in a boiling water bath or microwave oven. Note: If the agarose solution is heated in the microwave for a long time, the solution will overheat and bump. It should be checked whether the volume of the solution is reduced by evaporation during the boiling process and, if necessary, supplemented with buffer.
(4) Allow the solution to cool to 60 °C. Ethidium bromide (formulated with 10 mg/ml stock solution) was added to a final concentration of 0.5 ug/ml and thoroughly mixed.
(5) Pipette a small amount of agarose solution to seal the edge of the mold. After solidification, place the comb at a position of 0.5-10 mm from the bottom plate to form a good sample hole after adding agarose. If the comb is too close to the glass plate, there will be a risk of rupture at the bottom of the hole when the comb is pulled out, which will allow the sample to penetrate from the glass plate.
(6) Pour the remaining warm agarose solution into the mold. The thickness of the gel is between 3-5 mm. Check for any air bubbles under the teeth or between the teeth.
(7) After the gel is completely solidified (at room temperature for 30-45 minutes), the comb and the autoclaved tape are carefully removed, and the gel is placed in an electrophoresis tank.
The low melting point agarose gel and the agarose gel with a concentration below 0.5% should be cooled to 4 ° C and electrophoresed in a cold storage.
(8) Add enough electrophoresis buffer just about 1 mm deep on the rubber surface.
4. Sample loading
After mixing the DNA sample with the desired loading buffer, slowly add the mixture to the sample cell using a micropipette. At this point the gel has been immersed in the buffer. The maximum amount of a sample well to be added depends on the amount and size of the DNA, typically 20-30 μl of sample.
DNA standards of known size should be added to both the left and right wells of the left gel of the gel. Determine the size of the unknown DNA. When measuring the size of an unknown DNA, use the same sample buffer for all samples.
5. Electrophoresis
Under low voltage conditions, the migration speed of linear DNA fragments is proportional to the voltage, but as the electric field increases, the increase in the mobility of DNA fragments of different relative molecular masses is different. Therefore, as the voltage increases, the effective separation range of the agarose gel decreases. In order to obtain the maximum resolution of the electrophoretically separated DNA fragments, the electric field strength should not be higher than 5 V/cm. The electrophoresis was stopped when the bromophenol blue indicator was moved to about 1-2 cm from the lower edge of the rubber plate.
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