Hancock Lab

Southern Blot

Categorized: Bacterial Genetics

  1. Photograph ethidium bromide stained gel with a ruler.
  2. Soak gel in 0.25 M HCl for 10 minutes (21.5 ml concn HCl/l or 4.3 ml/200 ml)
  3. Soak gel in 1.5 M NaCl and 0.5 M NaOH for 30 minutes.
  4. Neutralize in 1 M Tris pH 8 and 1.5 M NaCl for 30 minutes.
  5. Set up transfer dish using 10X SSC.
  6. Invert gel and place onto 3MM paper.
  7. Mark nylon membrane with a pencil for later orientation and place onto gel; make sure there are no air bubbles between the gel and the nylon membrane.
  8. Put 2 layers of wet 3MM on top of the nylon membrane.
  9. Put 2 layers of dry 3MM on top of the wet 3MM paper.
  10. Let blot 18-24 hours for chromosomal DNA; 6-8 hours for other DNA (plasmid, etc.).
  11. Rinse blot in 2X SSC and UV 30-60 seconds on each side and bake at 80ºC for 2 hours.

NOTE:

If using positively charged nylon, following treatment with HCl, transfer with 0.4 M NaOH and forget about all the other stuff (i.e. use positively charged nylon if at all possible and it will save you a lot of time).

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