Northerns Dominik Haudenschild, March 6, 1998
Use RNAse free reagents and techniques
For 200ml
• To 162ml DEPC treated water add 2g RNAse free agarose
• Microwave to completely dissolve agarose
• Add 20ml 10x MESA buffer (Sigma#M5755)
• Add 18ml 37% Formaldehyde (to 1.1M)
• Pour gel in fume hood
For 50ml
• To 40.5ml DEPC treated water add 0.75g RNAse free agarose
• Microwave to completely dissolve agarose
• Add 5ml 10x MESA buffer
• Add 4.5ml Formaldehyde
• Pour gel in fume hood
Northern Blotting using Stratagene's PosiBlot
For formaldehyde gels
• Pretreatment
-Soak gel in 0.05N NaOH/0.15M NaCl for 20 minutes, shaking
• Neutralization
-Soak gel in 0.1M Tris pH 7.5/0.15M NaCl for 30 minutes
• Assemble PosiBlot apparatus as in Stratagene's instructions
• Blot for 30 to 60 minutes at 75mm Hg pressure
• UV crosslink the DNA onto membrane using Stratalinker on AutoCrosslink setting
For multiple reprobing, bake >2 hours at 80°C
• Visualize bands on gel with 0.04%Methylene blue in 0.3M Sodium Acetate pH 5.2
• Destain in water until background is almost white