Southern Blot

1. Run the gel as normal. Often for genomic Southerns, it is desirable to run long gels (18 cm) over 4–6 hrs.
2. Photograph the gel along with a ruler adjacent to the molecular weight markers as a reference.
3. Alkali transfer buffer = 0.5M NaOH (20 g/L), 1.5M NaCl (87.66 g/L). Prepare 1 liter for 1 gel and another 750 mL for each additional gel. Beware that this buffer is very dangerous, capable of causing severe eye damage. Use the large volumes involved in this procedure with care and wear protective glasses.
4. Add the gel to 250 mL alkali transfer buffer, plus 125 mL for each additional gel.
5. Place gel on rocker with buffer solution for 20 mins. Note that lowpercentage agarose gels must be agitated slowly to prevent tearing.
6. Keep the remaining 500 mL for the transfer tank.
7. Wear gloves for the following steps.
8. Cut 2 pieces of large 3-mm paper (wicks) and 2 pieces about 2-mm smaller than the gel on each edge and 1 piece of nylon (Hybond N⁺, Amersham) the same size as the gel.
9. Cut a stack of paper toweling about 2-mm smaller than the gel. The stack needs to be about 6-cm thick.
10. Prewet nylon in Double Distilled Water (DDW), then soak in alkali transfer buffer.
11. Add buffer to transfer tank to the level of the platform. Wet the long wicks in transfer buffer and place in the tank.
12. Place gel on platform and spoon on transfer buffer. Add the nylon and smooth out any bubbles with the back of your finger.
13. Add the 2 slightly smaller 3-mm filters, the first prewetted, the second dry.
14. Add the stack of paper towels. Note it is very important that the edges of the towel do not touch the wicks that the gel is sitting on or the transfer will be “shortcircuited”.
15. Top the stack with a glass or plastic plate and a weight (a bottle with about 200–300 mL H2O is ideal). Too much weight compresses the gel and terminates the transfer early.
16. Allow to transfer overnight and then remove the stack carefully. Mark the position of the wells on the filter with a biro (and note position of well #1) before removing the filter. Soak the filter in 0.5M Tris pH 7.5, 1.5M NaCl for 5 min after removal from the gel.
17. Add filter to 200 mL 2X SSC and allow the soak without agitation for 5 min.
18. Remove filter and blot dry and bake at 80 for 2 hrs or place in autoclave for 10 min when not operating but warm.
19. Prehybridge with 10 mL Aqua. hybridge (reagents), 1% SDS and 100 mg/mL boiled herring sperm DNA for 20–30 minutes (cloned DNA southern) to several hrs (genomic southern).
20. Boil probe for 3 min and add to 3 mL of fresh hybridge solution/SDS/ herring DNA. Squeeze prehybridge from the bag and add probe. Seal, avoiding air bubbles, and distribute the probe well. Incubate for 4–6 hrs (cloned DNA) to 16–20 hrs (genomic Southern). Washes are performed in 2 to 0.2X SSC, 0.1% SDS depending on the homology of the probe to target.