The hybridization of the Northern Blot Membrane
25 mL hybridization buffer (0.5 M sodium phosphate buffer pH 7.2
containing 7% SDS) and the rotating bottle were heated in a
hybridization oven (Hybrigene Techne FHB4DP, #3945507) at 62°C in
darkness. The nylon membrane was placed in the bottle and rotated for
two hours. The fluorescent-labelled DNA oligonucleotide was added at the
2 nM final concentration, and hybridization occurred overnight. The next
day, the wash buffer (20 mM sodium phosphate buffer pH 7.2 containing
1% SDS) was warmed in a water bath to 43°C. The membrane was washed
with 250 mL of pre-warmed wash buffer at 43°C thrice for 5 minutes on an
orbital shaker in a metal box, preventing light exposure. Finally, the
membrane was placed into a plastic envelope. The scanning of the
membrane was done in the Amersham Typhoon™ laser scanner.