Hybridization with Oligonucleotide Probes
1. Prepare Hyb solution. Plan for 0.2 ml/cm2. For mini-blot, try 10 mls. For larger blot, try 20
mls. Use tightly sealed plastic tray. Combine ingredients in the order listed.
Final Composition Northern To add
5X SSC 2.5 ml 20X SSC
ddH2O 6.8 ml ddH2O
2.5X Denhardt's 0.5 ml 50X Denhardt's
0.05% NaPPi 0.1 ml 5% NaPPi
0.1 mg/ml yeast tRNA 0.1 ml 10 mg/ml yeast tRNA
2. Northern: For glyoxal gels, incubate membrane in 20 mM Tris, pH 8 for 5 minutes at 65°C. Float
membrane in 5X SSC and submerge for 2 minutes to wet.
3. Place blot in HYB solution in sealable tray. Prehybridize at 37°C for 1 hour.
4. Remove 1 ml of HYB solution from tray, mix with labeled oligonucleotide probe, add back to tray.
5. Incubate overnight at 37°C.
6. Transfer filter to fresh tray containing 5X SSC/0.05% NaPPi at RT.
7. Gently agitate for 5 minutes at RT. Repeat wash twice more for 5 minutes each.
· Note for steps 7-9: Do not wash for longer than 5 minutes at each step. Short hybrids are not
very stable. Long wash times will reduce signal intensity.