Hoefer HB1000 - Instrukcja (Strona 27)

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p18
Procedure
1. Add 15 mL of prehybridization solution to each
hybridization bottle containing the blot. Remove
bubbles between the glass and blot. Cap the
bottles, attach the bottles to the wheels of the
bottle holder carousel, and close the hybridization
oven door.
2. Incubate the blot at 42 °C for 1 hour.
3. Remove the prehybridization solution and replace
with 10 mL of hybridization solution.
4. If using radiolabeled probes work safely from the
other side of a beta-blocking shield. Pipette 1 × 10 6
counts per minute of radiolabeled probe, or 200 ng
of biotinylated probe, into a 15 ml plastic tube.
Seal the tube with a plastic cap and poke a hole in
the top with a syringe needle to prevent pressure
build-up during boiling.
5. Denature the probe by placing the samples in the
boiling water bath and heating for 10 minutes.
Immediately transfer the tube to ice for 5 minutes
(to prevent renaturation). Add 5 mL of hybridiza-
tion buffer to the probe and transfer to the hybrid-
ization bottle containing the blot. AVOID pouring
the probe directly onto the blot.
6. Incubate in the Hoefer HB1000 Hybridization Oven
for 6 to 8 hours at 42 to 56 °C.
Washing the blot
• Tupperware container (sized to contain the blot)
• 0.1X SSC, 0.1% SDS (pre-warmed to 50 °C)
• 2X SSC, 0.1 % SDS (room temperature)
• 2X SSC (room temperature)
• 0.15X SSC, 0.1% SDS (pre-warmed to 50 °C)
• Gloves
• Filter paper
• Cardboard
• Plastic wrap
• Tape
For radioactive probes, you will also need:
• X-ray film holder
• X-ray film
• Intensifying screen