S&S TURBO BLOTTER

SOUTHERN BLOT:

1. Denaturation of DNA Gels

  1. Incubate the gel in denaturing buffer 2 times, 30 minutes each time. (Slowly shake tray containing gel.)

  2. Wash the gel in transfer buffer 15min.
2. Transfer of DNA in Gels to Nylon Membrane
  1. Using gloves, cut Nylon transfer membrane to gel size.

  2. Soak transfer membrane in distilled water for 15min.

  3. Place "stack tray" of TurboBlotter Rapid Downward Transfer Device on bench, making sure it is level.

  4. Place 20 sheets of dry GB004 blotting paper (thick) in stack tray.

  5. Place 4 sheets of dry GB002 blotting paper (thin) on top of stack.

  6. Prewet 1 sheet of GB002 blotting paper in transfer buffer on stack.

  7. Place wet transfer membrane on stack.

  8. Cover wet transfer membrane with agarose gel, making certain that there are no air bubbles between the gel and the membrane.

  9. Wet the top surface of the gel with transfer buffer and place 3 sheets of GB002 blotting paper, presoaked in transfer buffer on top of the gel.

  10. Attach the "buffer tray" of the transfer device to the bottom tray using the circular alignment buttons to align both trays.

  11. Fill the buffer tray with 125ml transfer buffer.

  12. Start the transfer by connecting the gel stack witht he buffer tray using the pre-cut "buffer wick" (included in each blotter stack), presoaked in transfer buffer. Place the wick across the stack so that the short dimension of the wick completely covers the blotting stack and both ends of the long dimension extend into the buffer tray. Place the "wick cover" on top of the stack to prevent evaporation. Make sure the edges of the wick are immersed in the transfer buffer.

  13. Continue the transfer for 1 hour. Additional transfer time may be needed if the gel is thicker than 4mm or with larger fragments of nucleic acids.
Neutralization

Following transfer, gently wash the transfer membrane in 1X neutralizing buffer.

Immobilization (Two choices are possible, here.)

1st Choice (Baking): Place membrane on fresh sheet of dry GB002 blotting paper and bake at 80 degrees C for 20min to 2 hours.

2nd Choice (UV Crosslinking of DNA to Membrane): Place membrane on fresh sheet of dry GB002 blotting paper to remove excess buffer and then expose damp membrane to the transilluminator for a total dose of 120mJ/cm2.

Storage

Blots treated as above may be stored at 4 degrees C for several months.

© Sonia Wallman, NHCTC. 1997-2000