UVP HybriCycler - Manual (Page 12)

HC-3000 HybriCycler Hybridization Oven 12
12. 1 mL DNA polymerase
13. Mix gently and incubate at 37°C for 1 hour
14. Stop the reaction by adding 2 mL EDTA
15. Store probes at -20°C in the dark.
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.
UVP Hybridization bottle(s) and caps
15 mL plastic tube
Boiling Water Bath
Bucket of ice
Glove
Plexiglass
shield
UVP Minidizer™, HybriCycler™, or Hybridizer™
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.