Hancock Lab

Blotting and Hybridization with Sodium Phosphate

Categorized: Bacterial Genetics

  1. Rinse blot with 2X SSC and air dry
  2. Place blot into hybridization tube with ~100 ml 0.5% SDS, 0.1 SSC. Prehybridize for 1 hour at 65C.
  3. Discard prehybridization solution and add hybridization buffer; use approximately 7-10 ml per large hybridization tube.
    25 ml 1.0 M NaHPO4
    200 µl 0.5 M EDTA
    50 ml formamide
    7 g SDS
    H2O to 100 ml
  4. Prehybridize for ~5 – 10 minutes at 37-42ºC.
  5. Add denatured and labeled probe.
  6. Hybridize 16 – 24 hours at 37-42ºC.
  7. Discard hybridization solution to radioactive waste. Wash the membrane briefly in 2X SSC, 0.1% SDS at 23ºC (room temperature).
  8. Wash the membrane in ~ 100 ml 0.1X SSC , 0.1% SDS for 15-30 minutes at 37-65ºC.
  9. Repeat step 8.
  10. Blot the membrane onto filter paper, wrap in plastic and expose to film.

Strip nylon membrane for reuse:

  1. Wash the blot twice in 100 ml 0.1X SSC, 0.1% SDS at 95ºC for 30 minutes.
  2. Wash the blot as in step 1 but at 23ºC.

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