UVP HB-500 Minidizer - Manual (Page 13)

HB-500 Minidizer Hybridization Oven 13
Protocol 2:
Hybridization to Nylon or Nitrocellulose
Hybridization to nylon or nitrocellulose membranes containing Nucleic Acid is accomplished by adding
single-stranded probe to the membranes that have been previously incubated with prehybridization solution.
The prehybridization and hybridization solutions both contain buffers designed to prevent adventitious
binding of the probe to the filters.
Reagents and Equipment
Prehybridization/hybridization solution [45% formamide, 5X SSPE (0.9 M NaCl, 50mM sodium
phosphate buffer, pH 7.4, 5mM EDTA), 0.1% SDS, 5X Denhardt’s solution (0.1% each of Ficoll,
polyvinylpyrrolidone, and bovine serum albumin), and 100 mg/mL of denatured salmon sperm DNA).
Mix well and remove aggregates before use.
Notes: When preparing prehybridization/hybridization solutions, add dry reagents directly to the
formamide/SSC solution. Incubate with mixing at 40-50°C for 2 hours or until dissolved. Store at 20°C.
SDS will precipitate at room temperature but remain in solution at 37°C.
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UVP Hybridization bottle(s) and caps
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15 mL plastic tube
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Boiling Water Bath
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Bucket of ice
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Gloves
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Plexiglas shield
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UVP Minidizer, HybriCycler, or Hybridizer Hybridization Oven
Procedure
1. Add 15 mL of prehybridization solution to each hybridization bottle containing the blot. Remove
bubbles between the glass and blot. Cap the blots and close the Hybridizer.
2. Incubate the blot at 42 °C for 1 hour.
3. Remove prehybridization solution and replace with 10 mL of hybridization solution.
4. Pipette 1x106 counts per minute of radio labeled probe or 200ng of biotinylated DNA 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
hybridization buffer to the probe and transfer to the hybridization bottle containing the blot: AVOID
pouring the probe directly onto the blot.
6. Incubate in the UVP HybriCycler, Hybridizer, or Minidizer 6 to 8 hours at 42 to 56 degrees.
Washing the Blot
•
Tupperware container (sized to contain the blot)
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0.1X SSC, 0.1% SDS (pre-warmed to 50
� C)
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2X SSC, 0.1 % SDS (room temperature)
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2X SSC (room temperature)
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0.15X SSC, 0.1% SDS (pre-warmed to 50
� C)
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Gloves
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Filter Paper
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Cardboard
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Plastic wrap
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Tape
Non-Radioactive Probes
1. Wash blots in 2X SSC, 0.1% SDS for 3 minutes at room temperature (repeat one)
2. Wash filter in 0.15X SSC, 0.1%SDS for 15 minutes at 50 °C (repeat once)
3. Store blots in 2X SSC at room temperature