4.12.2. Assembly of the transfer setup and transfer of RNA from gel to membrane

  1. To prepare a gel for transfer, rinse the gel in DEPC-treated water twice for 20 min to remove the formaldehyde, which will otherwise interfere with transfer of RNA from gel to the membrane.
  2. Soak the gel in RNase-free 20X SSC (3.0 M NaCl and 0.3 M sodium citrate, pH 7.0) for 45 min before proceed to setting up the transfer.
  3. Cut uncharged nitrocellulose membrane to size of gel.
  4. Soak the membrane in water for 2-3 min to wet .
  5. Float in 20X SSC.

The transfer is conducted by the capillary method (Fig. 2).

  1. Place a piece of thick blotting paper on the top of a glass plate that is        elevated by four rubber stoppers placed near each corner of a baking glass      dish.
  2. Drape the ends of the wick blotting paper over the edges of the plate.
  3. Fill the glass dish with RNase-free 20X SSC until the wick blotting paper on the top of glass plate is completely wet.
  4. Squeeze out all air bubbles by rolling with a glass rod or pipette.
  5. Place the gel facing down on the wet blotting paper.
  6. Squeeze out air bubbles by rolling a glass pipette.
  7. Cut a small triangular piece from the top left-hand corner to simplify orientation.
  8. Place the wetted membrane on the surface of the gel by aligning the cut corners.
  9. Get rid of any air bubbles under the membrane by rolling a glass pipette.
  10. Cut 4-5 sheets of Whatman 3MM paper to the same size as membrane.
  11. Place on top of the membrane.
  12. Place a stack of paper towels on top of the Whatman 3MM papers.
  13. Add a 200-500 g weight to hold everything in place.
  14. Allow the transfer of RNA to proceed by capillary action overnight.
  15. Disassemble the transfer stack at the next day.
  16. Rinse the membrane briefly in 6X SSC. 
  17. Immobilize RNA to the blot by UV cross linking while the membrane is still damp.

Fig. 2. Schematic diagram of the process used to transfer nucleic acids from a gel onto a binding membrane.

Figure 2