Summary: Southern Blotting - Alkaline Transfer to Positively Charged Nylon
1. Prepare and run a 0.75 - 1.25 % agarose gel 1 cm thick. Include DNA standards. Stain with
ethidium bromide. Photograph gel with ruler. Place gel in a clean glass dish.
2. Depurination: Treat gel with 5-10 volumes of 0.25 M HCl. Agitate gently at room temperature for
15 minutes. Pour off HCl and repeat treatment. Bromophenol blue dye will turn green/yellow.
0.25 M HCl = 22 ml concentrated HCl per liter
3. Rinse gel with dH2O.
4. Treat gel with 5-10 volumes of 0.4 M NaOH. Agitate gently at room temperature for 15 minutes.
Pour off NaOH and repeat treatment.
0.4 N NaOH = 16 g per liter (add pellets to dH2O with continuous stirring)
5. Set up transfer. Use 0.4 M NaOH as transfer solution. Use 2 pieces of Whatman 3MM paper to
make wick. Place wick over glass plate on support platform. Wet wick with NaOH. Put gel face
down on wet wick. Cut off bottom right corner of Nylon membrane for orientation. Pre-wet with
NaOH and place on gel. Remove air bubbles by gently rolling a glass test tube over membrane.
Place 4 pieces of pre-wetted Whatman 3 MM paper on top. Remove air bubbles again. Add a tall
stack of dry cut paper towels. Add ~100 g weight (glass plates work well). Avoid excess weight,
which will compress gel. Place plastic wrap around gel to prevent evaporation.
6. Alkaline capillary transfer is faster than high salt (SSC) transfer. Transfer is usually complete in 2
hours. Overnight transfer does not harm DNA or membrane.
7. Remove and discard paper towels and filter paper. Rinse membrane twice for 5 minutes in 2X SSC.