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Southern Transfer to Nylon Membrane in NaOH

来源:生物谷  2010/7/2   访问量:2546
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Principle:

    Refer to Southern Transfer with Zetabind membrane. The NaOH transfer may be a more efficient transfer method for larger sized fragments. It is the method of choice for pulsed-field gels containing fragments >= 20 kb. Since smaller sized fragments are not transferred as efficiently and because of the large volumes of NaOH involved, we do not use the procedure for routine Southern transfers.

Time required:

    Day 1: 2 hours
    Day 2: 3 hours

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 65癈 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):

  • Denaturing solution, per liter:

    0.5 N NaOH 50 ml 10N NaOH
    1.5 M NaCl 500 ml 3M NaCl
    deionized water 450 ml dH20

  • Wash solution, per liter:

    0.5 M Tris 500 ml 1M Tris-HCl
    1 M NaCl 333 ml 3M NaCl
    deionized water 167 ml dH20

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.

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