Agarose gel electrophoresis experimental procedure - Database & Sql Blog Articles

Agarose gel electrophoresis experimental procedure, first, material 1. Buffer and solution agarose solution running buffer (usually 1X TAE or 0.5X TBE) ethidium bromide or SYBR Gold staining solution 6X gel loading buffer 2. Nucleic acid and oligonucleotide DNA sample DNA size standard 3. Special equipment agarose gel electrophoresis instrument sealing tape microwave or boiling water bath can provide 500V and 200mA power supply unit 55 ° C water bath II, method 1. Sealed with edge band The open sides of the plastic tray or the edges of the clean, dry glass plate form a mold that is placed on a horizontal support. 2. Prepare a sufficient amount of running buffer (1X TAE or 0.5X TBE) to fill the electrophoresis tank and prepare the gel. 3. Prepare the appropriate concentration of agarose solution in the running buffer according to the size of the DNA fragment to be separated: The agarose dry powder should be accurately weighed into a conical flask or glass bottle containing a predetermined amount of running buffer. 4. Loosely lock the neck of the flask with Kimwipes. If it is covered with a glass bottle, it must be loosely covered. Heat in a boiling water bath or microwave until the agarose melts. 5. Transfer the flask or glass via a grommet or clip to a 55 ° C water bath. The gel to be melted was cooled slightly and then ethidium bromide was added to a final concentration of 0.5 μg/ml. Gently rotate to fully mix the gel solution. 6. When the agarose solution is cooling, use a suitable comb to form the well. The position of the comb teeth should be 0.5~1.0 mm on the bottom of the tray, so that the agarose will form a matching sample hole when it is poured into the tray. 7. Irrigate the warm agarose solution into the mold. 8. Allow the gel solution to completely condense at room temperature for 30 to 45 minutes. Add a small amount of running buffer to the top of the gel and carefully pull out the comb. Pour out the running buffer. Gently remove the edge banding tape and place the gel in the electrophoresis tank. 9. Add the running buffer to the electrophoresis tank, just before the gel is about 1 mm. 10. Mix the DNA sample and 0.2 volumes of 6X loading buffer. 11. Slowly add the sample mix to the immersion gel well with a micropipette and disposable tip, or an elongated Pasteur pipette, or glass capillary. Molecular mass standards should be added to the two wells on the left and right sides of the sample well. 12. Close the electrophoresis tank cover and connect the electrode plug. DNA should move toward the anode (red plug) side. A voltage of 1 to 5 V/cm is applied, wherein the distance is measured between the anode and the cathode. If the electrode plugs are properly connected, the anode and cathode will generate bubbles due to electrolysis, and bromophenol blue will migrate from the sample well into the gel within a few minutes. Electrophoresis was stopped after bromophenol blue and xylyl cyanide FF migrated to an appropriate distance. 13. When the DNA sample or dye has migrated a sufficient distance in the gel, turn off the power, pull out the electrode plug, and open the lid. If the gel and buffer are ethidium bromide, the gel is observed and photographed with an ultraviolet lamp. Otherwise, the gel is immersed in water containing ethidium bromide (0.5 μg/ml) or running buffer for 30-45 min at room temperature, or dilute with SYBR Gold stock solution diluted 1:10000 in running buffer.