Hoefer HB1000 - Instrukcja (Strona 26)

•
p17
Protocol
2
Hybridization to Nylon or Nitrocellulose
Hybridization of probe to target nucleic acid that
has been previously transferred to nylon or nitrocel-
lulose membranes is accomplished by adding single-
stranded probe to the membranes that have first been
incubated with prehybridization solution. Both the
prehybridization and hybridization solutions 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, 50 mM sodium
phosphate buffer, pH 7.4, 5 mM EDTA), 0.1% SDS,
5X Denhardt’s solution (0.1% each of Ficoll, poly-
vinylpyrrolidone, and bovine serum albumin), and
100 mg/mL of denatured salmon sperm DNA]. Mix
well and remove aggregates before use.
• Hoefer hybridization bottle(s) and caps
• 15 mL plastic tube
• Boiling water bath
• Bucket of ice
• Gloves
• Beta blocking shield
• Hoefer HB1000 Hybridization Oven
Note: 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.