3.3.2. Assembly of the transfer setup and transfer of DNA from gel to membrane

DNA is here pulled from the gel into a nylon membrane by capillary action pulled by the positive charge of the membrane. Once in contact with the membrane, DNA is attached using high-voltage cross-linking.

  1. Set up capillary transfer using 20X SSC as a transfer agent:
    Inside a baking glass dish filled with 20xSSC,place a glass plate that is elevated by four rubber stoppers that is slightly larger than the gel.
  2. Cover the glass plate with a piece of wick-blotting paper that has to be long enough so that it is in contact with the 20xSSC transfer solution.
    The buffer flows up the wick-blotting paper by capillary action, then through the gel to the membrane.
  3. Smooth out the air bubbles between the glass and the blotting paper by gently rolling with a glass pipette.
  4. Place the gel facing down on the wet blotting paper.
  5. Cut a small triangular piece from the top left-hand corner to simplify orientation.
  6. Smooth out the air bubbles.
  7. Cut one piece of positively charged nylon membrane to match size of the gel.
  8. Soak the membrane in water for 2-3 min to wet and then float in 20X SSC.
  9. Gently place the membrane on the top of the gel.
  10. Mark well positions on the membrane.
  11. Smooth out the air bubbles.
  12. Cut 4-5 sheets of Whatman 3MM paper to the same size as the gel and place on top of the membrane.
  13. Place a stack of paper towels on top of the Whatman 3MM papers.
  14. Add a 200-400 g weight to hold everything in place.
  15. Allow the DNA to transfer for 10-16 hours.
  16. After transfer, rinse the membrane briefly in 6X SSC.
  17. Immobilize DNA to the membrane by UV cross-linking (120,000 microjoules per cm²). Membrane is now ready for labelling (section 3.3.3.).