To perform southern blotting, start by cutting a nylon membrane into a 10 by 12 centimeter sized segment. Make a notch in the same position as the gel. Activate the membrane by dipping it in distilled water, followed by 20X sodium saline citrate buffer.
Place a filter paper wick on a double inverted tray, set such that the ends touch the base of the outer tray. Place the gel over the filter paper. Then place the activated nylon membrane over the gel, ensuring that the notches overlap.
Remove any air bubbles with a glass rod. Place a two centimeter heap of 9.5 by 11.5 centimeter sized cellulose filter paper, and another six centimeter heap of regular filter paper. Place a weight atop the setup for equal weight distribution.
Fill the outer tray with 20X sodium saline citrate buffer, and leave it overnight for efficient transfer. After southern transfer, fix the transferred DNA on the membrane by UV crosslinking on a UV transilluminator. Wash the membrane twice with 25 milliliters of 2X sodium saline citrate buffer.
To perform hybridization, incubate the membrane in 10 milliliters of the pre-warmed pre-hybridization buffer. Gently agitate the membrane manually at frequent intervals. Then incubate the blot in 10 milliliters of hybridization buffer at 42 degrees Celsius for three hours with gentle agitation.
Wash the blot twice in 25 milliliters of stringent buffer for 10 minutes at room temperature with gentle agitation. Now wash the blot twice in 25 milliliters of pre-warmed stringent buffer at 50 degrees Celsius for 15 minutes with gentle agitation. Rinse with 15 milliliters of wash buffer for five minutes with gentle agitation at room temperature.
Incubate the membrane in 10 milliliters of freshly prepared blocking solution for 30 minutes at room temperature with gentle agitation. Then place it in 10 milliliters of anti-digoxigenin conjugated with an alkaline phosphatase working solution. Wash the blot twice in the wash buffer at room temperature for 15 minutes.
Now incubate in 10 milliliters of detection buffer for five minutes at room temperature. After removing the excess buffer, place the membrane on an acetate sheet with the DNA side facing up. Add approximately one to 1.5 milliliters of substrate solution dropwise on the membrane.
Immediately place another acetate sheet on it and incubate for five minutes at room temperature. Squeeze out the excess substrate solution before imaging. Using a gel documentation imaging system, collect multiple unsaturated images at different time points for analysis.
After southern blotting and hybridization, the telomere restriction fragments were clearly visible, indicated by a smear.
