Agarose gel electrophoresis is the standard method for the isolation and identification of DNA fragments. This technique is simple and fast, and can resolve DNA fragments that cannot be separated by other methods such as density gradient centrifugation. When stained with a low concentration of fluorescent intercalating dye ethidium bromide EB (Deyuan International Cat# LY5009), at least 1-10 ng of DNA bands can be detected under ultraviolet light, so that the position of the DNA fragment in the gel can be determined. . In addition, specific DNA bands can be recovered from the gel after electrophoresis for subsequent cloning operations. The agarose gel separates a wide range of DNA fragments, and different concentrations of agarose gel can separate DNA fragments from 200 bp to nearly 50 kb in length. Agarose is usually electrophoresed using a horizontal device under an electric field of constant intensity and direction. The logarithm of DNA electrophoretic mobility is linear with gel concentration. The choice of gel concentration depends on the size of the DNA molecule. The concentration of the gel required to isolate a DNA fragment of less than 0.5 kb is 1.2-1.5%, the concentration of the gel required to isolate a DNA molecule larger than 10 kb is 0.3-0.7%, and the concentration of the DNA fragment between the two is 0.8- 1.0%. First, the material DNA samples, agarose, buffers, etc. Second, the equipment Horizontal electrophoresis apparatus, electrophoresis apparatus, benchtop high speed centrifuge, constant temperature water bath, micropipette, microwave oven or electric furnace, UV transilluminator, photo holder, camera and accessories. Third, the reagent 1, 5 × TBE electrophoresis buffer (Deyuan International Cat# LY5010): 2, 6 × electrophoresis sample buffer (Deyuan International Cat# LY50013): 3, ethidium bromide (EB) solution mother liquor: EB is formulated into 10mg / ml, wrapped in aluminum foil or black paper, stored at room temperature. Fourth, the preparation of agarose gel 1. Take 20ml of 5×TBE buffer and add water to 200ml, and prepare 0.5×TBE dilution buffer for use. 2, the preparation of the glue: weigh 0.4g agarose, placed in a 200ml Erlenmeyer flask, add 50ml 0.5 × TBE dilution buffer, put it in a microwave oven (or electric stove) heated until the agarose is completely melted, remove and shake This is a 0.8% agarose gel solution. Shake from time to time during the heating process to allow the agarose particles attached to the wall of the bottle to enter the solution. The sealing film should be covered when heating to reduce evaporation of water. 3. Glue: Put the organic tray into the glue mold and insert the sample comb. Observe that the lower edge of the comb tooth should be kept at a distance of about 1mm from the bottom surface of the glue groove. Add an ethidium bromide (EB) solution to the agarose gel cooled to 50-60 ° C to a final concentration of 0.5 μg / ml (you can also add EB to the gel instead of 0.5 μg / after electrophoresis) Mol EB solution soaked for staining). Carefully pour the agarose into the tank to form a uniform layer of glue. The temperature when pouring the glue should not be too low, otherwise the solidification will be uneven, and the speed should not be too fast, otherwise bubbles will easily appear. After the gel is completely solidified, the comb is pulled out, taking care not to damage the gel at the bottom of the comb, and then adding 0.5×TBE dilution buffer to the tank to the surface just before the surface of the rubber plate. Due to the edge effect, there will be some bulges near the sample tank, which will prevent the buffer from entering the sample tank, so care should be taken to ensure that the sample tank is filled with buffer. 4. Loading: Mix 10 μl of the enzymatic hydrolysate with 2 μl of 6× loading solution and carefully add to the sample tank with a micropipette. If the DNA content is low, the sample loading can be increased according to the above ratio, but the total volume cannot exceed the sample tank capacity. Replace the tip head every time you add one sample to prevent mutual contamination. Be careful when handling the sample to avoid damaging the gel or piercing the gel at the bottom of the sample tank. 5. Electrophoresis: After adding the sample, close the electrophoresis tank cover and immediately turn on the power. The control voltage is maintained at 60-80V and the current is above 40mA. Electrophoresis was stopped when the bromophenol blue band moved about 2 cm from the gel front. 6. Staining: The gel plate without EB was transferred to 0.5 μg/ml EB solution after electrophoresis, and stained at room temperature for 20-25 minutes. 7. Observation and photographing: The electrophoretic rubber sheet after dyeing or having EB was observed under a long-wavelength ultraviolet lamp with a wavelength of 254 nm. The presence of DNA shows an orange-red fluorescent band that is visible to the naked eye. Wear protective glasses or a plexiglass mask when viewing under a violet light to avoid eye damage. After attaching the close-up lens and red filter to the camera lens, the camera is fixed to the photo frame, using full-color film, aperture 5.6, and exposure time of 10-120 seconds (according to the depth of the fluorescent strip).
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