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Filter hybridization is a method used to qualitatively determine the presence of homologous or complementary sequences in DNA or RNA, by allowing a denatured radio-labeled 'probe' nucleic acid (in solution) to anneal to the denatured nucleic acid (immobilized to a nitrocellulose filter) to be tested. The filters containing the immolbilized nucleic acid can be Southern blots, Northern blots, lifted colonies or plaques, or spot filters.
For Northern blots or RNA probes, use strict RNase-free technique. For DNA-DNA hybridizations, poly(A) and blocking DNA may not be needed. At low stringency, blocking DNA may interfere with proper hybridization. Choose a blocking DNA that is most dissimilar to both DNAs being hybridized (i.e. herring sperm DNA for hybridizing procaryotic DNAs, or E. coli DNA for hybridizing yeast against soybean). Stringency can be adjusted from high (68C) to low (down to about 50C) by lowering the temperature. Hybridizations at high stringency, as described here, with DNAs of perfect homology, will require about 100 KCPM of 32P-probe DNA. Hybridizations at low stringency generally require more probe CPM.

  1. Materials

    1. 20X SET
      Final Conc.
      175.3 grams NaCl 3.0M
      72.6 grams Tris-OH 0.6M
      14.9 grams Na2EDTA 40mM
      1 liter
      Adjust to pH 8.0 with HCL

    2. 50X Denhardts
      Final Conc.
      1.0 gram BSA 1%
      1.0 gram Ficoll 1%
      1.0 gram PVP-360 1%
      100ml

    3. 20% SDS
      10 grams per 50ml

    4. 10% Na pyrophosphate
      5 grams per 50ml (boil to dissolve)

    5. 10 mg/ml poly(A)
      10 mg plus 1.0ml ddH2O (store -20C)

    6. 50ug/ml sonicated herring sperm DNA
      5 mg plus 5ml ddH2O, stored at -20C and sonicated (Micro-tip, 3-5 min. at max frequency) in 1ml aliquots before use.

    7. Sol'n A (per 200ml)
      Final Conc.
      40ml 20X SET 4X
      160ml ddH2O

    8. Sol'n B (per 100ml)
      Final Conc.
      20ml 20X SET 4X
      20ml 50X Denhardts 10X
      0.5ml 20% SDS 0.1%
      59.5ml ddH2O

    9. Sol'n C (per 50ml)
      Final Conc.
      10ml 20X SET 4X
      10ml 50X Denhardts 10X
      0.25ml 20% SDS 0.1%
      50ul 10mg/ml poly(A) 10ug/ml
      0.5ml 10% Na pyrophosphate 0.1%
      28.7ml ddH2O

    10. Sol'n D (per 100ml)
      Final Conc.
      20ml 20X SET 4X
      20ml 50X Denhardts 10X
      0.5ml 20% SDS 0.1%
      1.0ml 10% Na pyrophosphate 0.1%
      100ul 10mg/ml poly(A) 10ug/ml
      58.5ml ddH2O

    11. Sol'n E (per 300ml)
      Final Conc.
      45ml 20X SET 3X
      1.5ml 20% SDS 0.1%
      3.0ml 10% Na pyrophosphate 0.1%
      250.5ml ddH2O

    12. Sol'n F (per 500ml)
      Final Conc.
      25ml 20X SET 1X
      2.5ml 20% SDS 0.1%
      5ml 10% Na pyrophosphate 0.1%
      468ml ddH2O

    13. Sol'n G (per 200ml)
      Final Conc.
      10ml 20X SET 1X
      190ml ddH2O

    14. Handle the filters with gloves!

  2. Methods

    1. Wet the filter by floating, then sinking it, in 200ml sol'n A at room temperature. Let the filter soak for 30 min.
    2. Transfer the filter to a zip-lock bag and add 100ml of sol'n B. Incubate with gentle shaking at 68C in a pre-warmed glass dish about half filled with water.
    3. Decant off the sol'n B. Add 25ml pre-warmed sol'n C and 0.25ml freshly denatured (boiling water bath, 3-5 min.) 10mg/ml sonicated herring sperm DNA. Incubate at 68C for at least 1 hr with gentle shaking.
    4. Decant off the sol'n C. Add 25ml pre-warmed sol'n C, 0.25ml freshly denatured 10mg/ml sonicated herring sperm DNA, and an appropriate amount of freshly denatured 32P-probe DNA. Incubate at 68C for at least 1 hr (usually overnight) with gentle shaking.
    5. Decant off the sol'n C in a liquid radioactive disposal container. Add 100ml pre-warmed sol'n D, and incubate at 68C for 1 hr with gentle shaking.
    6. Decant off sol'n D in a radioactive sink and add 100ml pre-warmed sol'n E. Incubate at 68C for 30 min. with gentle shaking. Repeat twice more.
    7. Decant off sol'n E into a radioactive sink and add 100ml pre-warmed sol'n F. Incubate at 68C for 30 min. with gentle shaking. Repeat once more.
    8. Decant off sol'n F into a radioactive sink and transfer the filter to a dish containing 100ml of room temperature sol'n G. Incubate at room temperature for 15 min. with occaisional agitation.
    9. Take the filter out and briefly lay it onto a sheet of Whatman #1 filter paper. Tape the filter onto a clean, heavy, 8 1/2 x 11 sheet of paper and cover with a single thickness of saran-wrap. The filters are now ready for autoradiography.

Maniatis, et al. 1978 Cell 15:687-701
Vaughn,J. (personal communication)

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