4.12.4. Hybridization analysis
(Roche Applied Science DIG Easy® Hyb, DIG Wash and Block Buffer Set, CSPD®Ready-to-use protocol)
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.
- Pipette the desired volume of probe (50-100 ng probe per ml hybridization buffer) into the hybridization bag.
- Continue to incubate with rotation at 65°C for 10-16 hours.
- 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.
- Transfer the blot in a preheat High Stringency Buffer (0.1x SSC containing 0.1% SDS).
- Incubate the blot twice (2 x 15 min, with shaking) in High Stringency Buffer at 65° C.
- After last wash, pull out the blot out of the hybridization container.
- 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.
- Place blot onto a piece of Plastic wrap that is at least twice the size of the membrane.
- 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.
- Completely wrap up the blot with the plastic wrap.
- Put it in a film cassette for chemiluminescent detection of hybridization signals.