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Method: Southern Transfer to Nylon Membrane in NaOH

4/18/1990

C. Helms


Principle:

Time required:

Procedure:

Day 1

Pretreatment:

  1. After staining and photographing the gel, denature by soaking in 500 ml or more of denaturing solution (0.5 N NaOH, 1.5 M NaCl) for 30 minutes with agitation.

Transfer:

  1. The transfer is done in a pyrex baking dish containing 4-6 blot blocks (BRL #1964BW, 20 x 25 cm). The blocks are saturated and the tray filled with fresh denaturing solution to the top of the stack of blot blocks. Two pieces of 3MM paper (cut to the size of the blocks) are wettedin the denaturing solution and placed on top of the blocks.
  2. Position the gel on top, and place a wicking barrier (Saran wrap) around the gel.
  3. Cut the Zetabind membrane slightly larger than the size of the gel. Label the membrane, prewet in deionized water, then place on the gel. Follow with 2 pieces of 3MM paper presoaked in denaturing solution. Be sure to eliminate air bubbles caught between layers (use a test tube rolled gently over the surface of each layer as the stack is constructed).
  4. Add 10 blot blocks to the top of the stack followed by paper towels. Allow the transfer to continue until all the denaturing solution is absorbed from the tray (usually overnight).

Day 2

Washing and baking the membrane:

  1. Wash the membrane in 0.5 N NaOH-1.5 M NaCl for 30 minutes with agitation.
  2. Wash twice in 0.5 M Tris-HCl pH7.5, 1 M NaCl for 30 minutes each wash; this step is essential to prevent high background.
  3. Wash in 10X SSC for 10 to 30 minutes. Air dry.
  4. Bake the blot for 1 hour at 80 degrees C between 2 pieces of blotting paper.

Prewashing:

  1. All new blots should be wetted in 2X SSC and then washed in 0.2X SSC, 0.2 % SDS at 65C for one hour to minimize background during hybridizations. The membrane can be stored in a sealed bag or prehybridized as usual.

Solutions (non- stock items):

References:

Sambrook, J., Fritsch, E.F., and T. Maniatis.(1989) Molecular Cloning, A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press. pp 6.24-6.27.

CRI Laboratory Methods Manual: RFLPs Project (1989), p 39.