4.12.2. Assembly of the transfer setup and transfer of RNA from gel to membrane
- 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.
- 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.
- Cut uncharged nitrocellulose membrane to size of gel.
- Soak the membrane in water for 2-3 min to wet .
- Float in
The transfer is conducted by the capillary method (Fig. 2).
- 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.
- Drape the ends of the wick blotting paper over the edges of the plate.
- Fill the glass dish with RNase-free 20X SSC until the wick blotting paper on the top of glass plate is completely wet.
- Squeeze out all air bubbles by rolling with a glass rod or pipette.
- Place the gel facing down on the wet blotting paper.
- Squeeze out air bubbles by rolling a glass pipette.
- Cut a small triangular piece from the top left-hand corner to simplify orientation.
- Place the wetted membrane on the surface of the gel by aligning the cut corners.
- Get rid of any air bubbles under the membrane by rolling a glass pipette.
- Cut 4-5 sheets of Whatman 3MM paper to the same size as membrane.
- Place on top of the membrane.
- Place a stack of paper towels on top of the Whatman 3MM papers.
- Add a 200-500 g weight to hold everything in place.
- Allow the transfer of RNA to proceed by capillary action overnight.
- Disassemble the transfer stack at the next day.
- Rinse the membrane briefly in 6X SSC.
- 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.