To begin, place the reagents required for the preparation of gel electrophoresis on a working platform. Add Tris/Borate/EDTA, or TBE, to agarose and heat the solution in a microwave. Then, pour the 0.4-0.5%agarose into a casting tray to prepare a first-dimension agarose gel and let it solidify for one hour.
After solidification, load the ladder within the first 3 centimeters relative to the gel's leftmost edge. Then load the DNA samples digested with restriction enzymes, ensuring a 3 centimeter distance between each pair. Run the gel in TBE for 19-24 hours at 0.85 volts per centimeter to separate the intermediates by size.
The following day, remove the gel from the buffer. Excise the first 3 centimeters of the gel containing the ladder. Stain the gel segment in TBE containing 0.3 micrograms per milliliter of ethidium bromide for 10-15 minutes.
Then visualize the ladder using a gel documentation system. On the agarose gel, add 1.3 centimeters to the estimated location, yielding a value of A.Then subtract 7.5 centimeters from value A, yielding value B.Align the ruler against the first-dimension gel and cut horizontally across at values A and B.Then cut vertically down the 3 centimeter space reserved for each sample. In a new casting tray, rotate the segments clockwise and place them in the position of the sample wells.
Prepare the second-dimension agarose gel at a concentration of 1-1.3%in TBE with 0.3 micrograms per milliliter of ethidium bromide. Heat the gel, and upon cooling to approximately 55 degrees Celsius, pour it over the rotated first-dimension segments. Allow the gel to solidify for one hour.
After solidification, transfer the gel to a chamber containing TBE at 0.3 micrograms per milliliter of ethidium bromide, and allow it to equilibrate for 30 minutes. Cover the gel and run for 9-10 hours at 4.23 volts per centimeter at 4 degrees Celsius. Remove the second-dimension gel from the chamber and depurinate the DNA fragments for 10 minutes in a 0.24 molar hydrochloric acid solution with gentle rocking.
Rinse the gel with deionized water and soak it in 0.4 molar sodium hydroxide for 10-15 minutes. Then fold two long sheets of chromatography paper perpendicular across the glass sheet, extending into the container. To assemble the southern blot, fill a container with 1 liter of 0.4 molar sodium hydroxide.
Align a long glass sheet across the container. Wet the top of the paper with sodium hydroxide and carefully remove any air bubbles under its surface. Soak three sheets of chromatography paper with sodium hydroxide and place them over the folder paper.
Remove any air bubbles. Flip the second-dimension gel upside down and place it over the chromatography papers. Wet a positively charged nylon membrane with deionized water and place it over the gel.
Then place three chromatography sheets wet with deionized water over the membrane. Cover any exposed sodium hydroxide in the container with plastic wrap to prevent evaporation. Place a 0.3-0.5 meter tall stack of napkins or paper towels over the blot.
Compress the entire blot with a weight to facilitate tight capillary action, and leave it for two days for DNA transfer to the membrane.
