Precautions and operation procedures of agarose gel electrophoresis
Precautions and operation procedures of agarose gel electrophoresis
Matters needing attention in gel electrophoresis:
1. Buffer system: In the absence of ions, the conductivity is the smallest, the DNA does not migrate, or the migration is extremely slow. In a buffer with high ionic strength, the conductivity is high and heat is generated, which may cause DNA denaturation, so you should pay attention to the buffer Is used correctly. For long-term high-pressure electrophoresis, it is advisable to renew the buffer or circulate the buffer between the two tanks.
2. Agarose: Different manufacturers and different batches of agarose have different impurity content, which affects the migration of DNA and the intensity of the fluorescent background, and should be used selectively.
3. Preparation of gel: The buffer added in the gel should be consistent with that in the electrophoresis tank. The dissolved gel should be poured into the plate in time to avoid coagulation and agglomeration before pouring. The gel poured into the plate should avoid air bubbles and affect the electrophoresis results.
4. Amount of sample added: In general, a 0.5cm wide comb can add 0.5ug of DNA. The amount of sample added depends on the size of the sample hole and the number and size of fragments in the DNA. This causes overloading of the sample well, which leads to tailing and dispersion, which is more pronounced for larger DNA.
5. Changes in the electrophoresis system will affect the migration of DNA, and it is necessary to add a DNA standard reference for judgment.
6. The salt concentration in the DNA sample will affect the DNA mobility. The parallel buffer samples should use the same buffer conditions to eliminate this effect.
7. DNA mobility depends on the concentration of agarose gel, the shape and size of the migrating molecules. It is possible to distinguish a wide range of DNA molecules using gels of different concentrations. The concentration of a gel can be determined according to the range of DNA molecules when preparing an agarose gel. The DNA electrophoresis of small fragments should use polyacrylamide gel electrophoresis to improve resolution.
Points to note when adding agarose:
1. Use pipetting to add the sample to the spot. The volume of each spot is generally less than 25ul, so when drawing each sample, the operation should be stable and careful.
2. A certain amount of sucrose is often added to increase the concentration of the sample, so that each sample stays in its own spot.
3. Add a water-soluble anion tracking dye (such as bromophenol blue) to the sample to see how far the sample moves.
4. Add standard molecular mass samples to one or several wells. After electrophoresis, the corresponding position of the band with known molecular mass can be used to make a standard curve.
5. Electrophoresis is generally stopped when tracking the dye swimming to 80% of the gel. Note that during electrophoresis, the cover of the electrophoresis tank should be securely covered to prevent evaporation of the liquid and reduce the possibility of electric shock.
6. After the electrophoresis is completed, immerse the gel in 1 mg / L ethidium bromide (EB). After 5 minutes, you can see the DNA band. EB is combined with NDA by inserting between the paired nucleotides in the double helix. Another method is to add EB to the gel during electrophoresis.
7. Under the ultraviolet lamp, the DNA band can be seen because the EB emits a strong orange-red fluorescence. The limit of detection using this method is approximately 10 ng DNA per band. Wear plastic safety glasses to prevent ultraviolet light from damaging your eyes. A ruler can be used to measure the distance from each strip to the spot. Similarly, using a special camera and focus adjuster, you can also take pictures of the gel.
8. If you want to further analyze a band (such as a plasmid), you can use a knife to cut the gel containing the band and recover the DNA from the band.
Operation procedure of agarose nucleic acid electrophoresis experiment
1. Rinse the rubber mold and comb with distilled water, put it on the rubber plate, close the edge of the mold, and set up the comb;
2. According to the size of the DNA fragments to be separated, prepare an appropriate concentration of agarose gel with gel buffer: accurately weigh the dry powder of agarose, add it to the triangle flask used for dispensing, and quantitatively add electrophoresis buffer (generally 20 ~ 30 ml) );
3. Put it in a microwave oven and heat it to melt it. After cooling for a while, add a drop of fluorescent dye, gently rotate to fully mix the gel solution, pour into the electrophoresis tank, and wait for it to solidify;
4. After 30 ~ 45 minutes at room temperature, the gel is completely coagulated. Carefully pull out the comb and place the gel in the electrophoresis tank;
5. Pour the electrophoresis buffer into the electrophoresis tank, the amount of which should be less than 1mm across the gel surface. If there are bubbles in the sample well, try to remove it
6. Add 10 × volume of loading buffer to the DNA sample. After mixing, use a gun to slowly add the sample mixture to the submerged gel sample well;
7. Turn on the power, red is the positive electrode and black is the negative electrode. Remember to run the DNA sample from the negative electrode to the positive electrode (the end near the sample hole is negative). Generally 60 ~ 100V voltage, electrophoresis can be 20 ~ 40min;
8. According to the position of the indicator swimming, determine whether to terminate the electrophoresis;
9. After electrophoresis is completed, turn off the power, observe the electrophoresis band and its position on the gel imager, and compare the size of the amplified product with the nucleic acid molecular weight standard Marker.
The best resolution range of agarose gel concentration and linear DNA The best resolution range of agarose gel concentration linear DNA (bp)
0.5% 1,000 ～ 30,000
0.7% 800 ～ 12,000
1.0% 500 ～ 10,000
1.2% 400 ～ 7,000
1.5% 200 ～ 3,000
2.0% 50 ～ 2,000
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