4.12.4. Hybridization analysis

(Roche Applied Science DIG Easy® Hyb, DIG Wash and Block Buffer Set, CSPD®Ready-to-use protocol)

  1. Pre-hybridize the blot with pre-warmed DIG Easy® Hyb (10– 15 ml per 100 cm2) in a specialized hybridization bag or any sealable container:
    1. Incubate the blot for 30 min at 65° C.
    2. Agitate gently during the pre-hybridization step.
  2. Pipette the desired volume of probe (50-100 ng probe per ml hybridization buffer) into the hybridization bag.
  3. Continue to incubate with rotation at 65°C for 10-16 hours.
  4. After the hybridization is complete, wash the blot in a tray containing Low Stringency Buffer (2x SSC containing 0.1% SDS) twice by incubating the tray at RT for 5 min with gentle agitation.
  5. Transfer the blot in a preheat High Stringency Buffer (0.1x SSC containing 0.1% SDS).
  6. Incubate the blot twice (2 x 15 min, with shaking) in High Stringency Buffer at 65° C.
  7. After last wash, pull out the blot out of the hybridization container.
  8. Place it between two Whatman paper sheets.
    Do not allow the membrane get too dry so the membrane can be stripped and reused for hybridization.
  9. Place blot onto a piece of Plastic wrap that is at least twice the size of the membrane.
  10. Add 1 ml detection reagent (anti-digoxigenin-AP conjugate and the premixed stock solution of CSPD® ready-to-use) to stain the membrane and leave for 5 min.
  11. Completely wrap up the blot with the plastic wrap.
  12. Put it in a film cassette for chemiluminescent detection of hybridization signals.