Agarose gel electrophoresis experiment
Agarose gel electrophoresis experiment
Agarose gel electrophoresis can be applied to (1) DNA cutting and recovery, (2) DNA isolation, (3) corroboration of whether DNA is recombinant or not, whether plasmids, etc. are cut or not, as well as other molecular biology studies.
Operation method
agarose gel electrophoresis
Principle
It is a kind of electrophoresis method using agarose as support medium. The main difference between its analyzing principle and other support electrophoresis is that it has the dual roles of "molecular sieve" and "electrophoresis". Agarose gel has a network structure, material molecules will be resisted when passing through, large molecules in the surge of material resistance, so in gel electrophoresis, the separation of charged particles depends not only on the nature and number of net charge, but also depends on the size of the molecule, which greatly improves the ability to discriminate. However, because of their rather large pore size, their molecular sieving effect is negligible for most proteins, and they are now widely used in the study of nucleic acids. Proteins and nucleic acids will have different charges depending on the pH, and will run at different speeds due to different magnitudes of force in the electric field, and can be separated according to this principle. The pH of electrophoresis buffer is between 6 - 9 and the ionic strength 0.02 - 0.05 is optimal. 1% agarose is commonly used as an electrophoretic support. Agarose gel can distinguish DNA fragments with a difference of about 100 bp, and its resolution is lower than that of polyacrylamide gel, but it is easy to prepare and has a wide separation range. Ordinary agarose gel separates DNA in the range of 0.2-20 kb, and using pulse electrophoresis, DNA fragments of up to 107 bp can be separated.DNA molecules have a charge effect and a molecular sieve effect when they swim in agarose gel.DNA molecules are negatively charged in pH solutions above the isoelectric point, and they move toward the positive pole in an electric field. Due to the structurally repetitive nature of the sugar-phosphate backbone, the same amount of double-stranded DNA has an almost equal net charge, so they can move toward the positive pole at the same rate.
Materials and Instruments
DNA Move I. Reagent preparation 1. 5×TBE buffer: 450 mmol/L Tris-boronic acid, 10 mmol/L EDTA, pH 8.0. 10-fold dilution of the above storage solution to 0.5×TBE buffer, which can be used as the buffer for electrophoresis and gel production at the same time. Gluing 1. According to the amount of gel production and gel concentration, in the triangular conical flask with a certain amount of electrophoresis buffer, add accurately weighed agarose powder (the total amount of liquid should not exceed 50% of the capacity of the conical flask). 2. 2. Cover the mouth of the conical flask with cling film or kraft paper and tie some small holes in the film or paper, and then dissolve the agarose by heating in a microwave oven. When the solution comes to a boil, wear heat-resistant gloves and carefully shake the conical flask to fully and evenly dissolve the agarose. Repeat this operation several times until the agarose is completely dissolved. 3. Allow the solution to cool to about 50°C-60°C. Add ethidium bromide solution (final concentration 0.5 μg/ml) or DNAgreen dye at a ratio of 1 μL / 30 mL if necessary, and mix well. 4. Pour the agarose solution into the gel-making mold and insert a comb at the appropriate position. The thickness of the gel is usually between 3 and 5 mm. Gel mold as shown in the figure below, the small gel poured into the agarose solution of about 25-30 mL, the large gel is about 60-70 mL, if you need to cut the gel recycling, the gel can be appropriately thickened. 5. Solidify the gel at room temperature, about 30 minutes ~ 1 hour. Third, on the sample 1. Take an appropriate amount of sample and 6×sampling buffer and mix well (e.g., 1 μl of sample and 5 μl of 6×sampling buffer), and add it into the sample tank carefully with a micropipette gun. 2. 2. The sample volume can be adjusted according to the concentration of the sample. If the DNA content is low, the sample volume can be increased according to the above ratio, but the total volume should not exceed the capacity of the sample tank (generally, the upper limit is 40 μl for small wells, and the upper limit is 200 μl for large wells, which is related to the specifications of the membrane.) 3. Replace the tip of the gun after each sample is added to prevent mutual contamination, and pay attention to careful operation when loading samples to avoid damaging the gel or puncturing the gel at the bottom of the sample tank. Electrophoresis 1. After adding samples, close the electrophoresis tank cover and turn on the power immediately. Keep the control voltage at 110 V and the current above 40 mA. 2. 2. When the strip moves to about 2 cm from the front edge of the gel (about 40 min), stop electrophoresis. 3. 3. Take pictures and observe under UV. Caveat 1. 5×TBE buffer will precipitate if left for too long, so do not mix too much at one time. The working electrophoresis buffer is 0.5×TBE buffer, take 5×TBE buffer storage solution to dilute, and use it now. 2. The buffer used for electrophoresis and the buffer used for glue making must be unified. 3. Agarose powder should not be heated in the microwave oven for too long, and stop heating every time when the solution bubbles and boils, otherwise it will cause the solution to overheat and boil violently, resulting in inaccurate concentration of the agarose gel, and will also damage the microwave oven. When dissolving agarose, it must be ensured that the agarose is fully and completely dissolved, otherwise, it will cause the electrophoresis image to be blurred. 4. When the gel is not used immediately, please wrap the gel with plastic wrap and store it at 4℃, generally it can be stored for 2~5 days. Common Problems Agarose gel concentration and optimal resolution range for linear DNA Agarose concentration Optimal resolution range of linear DNA (bp) 0.50% 1,000～30,000 0.7% 800～12 000 1% 500 to 10,000 1.20% 400 to 7,000 1.50% 200 to 3,000 2% 50 to 2,000 2% to 5% 20 to 1,000 For more product details, please visit Aladdin Scientific website.
Agarose TBE electrophoresis buffer Electrophoresis sample loading buffer Ethidium bromide (EB) solution mother liquor DNAGreen
Horizontal Electrophoresis Unit Electrophoresis Instrument Benchtop High Speed Centrifuge Constant Temperature Water Bath Micron Pipette Gun Microwave Oven or Electric Oven Ultraviolet Transmittance Instrument Photographic Stands Cameras and Their Accessories