Add water and heat to melt, load the comb, add the color developer at the right temperature (or run through the electrophoresis staining), and pour the gel plate.
The following is the operation of agarose gel electrophoresis:
1 Choose a suitable horizontal electrophoresis tank and adjust the plane of electrophoresis tank to horizontal. Check the lines of voltage stabilized power supply and positive and negative poles;
2 Select a spotting comb with appropriate aperture size, set it vertically on one end of the electrophoresis gel mold, and make the distance of the bottom of the spotting comb from the bottom of the electrophoresis gel mold 1.0mm;
3 According to the DNA to be separated, prepare the corresponding concentration of agarose, and heat it in a water bath at 100℃ until the agarose melts uniformly;
4 Take a small amount of the agarose gel solution by pipette. Seal the electrophoresis gel mold around the agarose gel solution with a pipette to prevent infiltration when pouring the agarose gel plate. When the agarose gel cooled down to about 50℃, add 6μl of nucleic acid dye, shake well, and gently pour the agarose gel into the electrophoresis gel mold, the thickness of the agarose gel was 3~5mm. when pouring the agarose gel, avoid air bubbles, and if there were air bubbles, use the pipette to suck them out carefully;
5 After the agarose gel solidified, leave it at room temperature for 20min, carefully pull out the spotting combs and the baffles on the ends of the electrophoresis gel molds, and keep the spots intact;
5 The spotting holes of the gel mold were sealed with a small amount of agarose gel solution and sealed around the electrophoresis gel mold.
6 Put the electrophoresis gel mold into the electrophoresis tank, add electrophoresis buffer, make the electrophoresis buffer surface 1~2mm above the surface of the agarose gel. if there are air bubbles in the spotting hole, use a pipette to suck them out carefully, so as not to affect the addition of samples;
7 Mix 10 μl of DNA samples with 2 μl volume of Bromophenol Blue Indicator Spotting Buffer. Sampling buffer can not only increase the density of the sample and make the sample sink evenly into the sample wells, but also make the sample with color, which is convenient for sampling and estimating the electrophoresis time and judging the position of electrophoresis;
8 Carefully add the sample into the spiking wells with the micropipettor, and record the order of the sample order of spotting;
9 Cover the electrophoresis tank and turn on the power switch, and the maximum voltage should not be more than 5V/cm ( 100～150V constant voltage electrophoresis), so that the DNA moves from the negative pole to the positive pole;
10 Electrophoresis time varies with the specific requirements of the experiment. Electrophoresis generally takes 1 to 3 hours. After electrophoresis is completed, turn off the power, wear disposable plastic gloves to remove the gel, drench all the electrophoresis buffer as much as possible, and observe it under the transmission ultraviolet lamp at 254 nm wavelength.