Northerns

NorthernsStuff you need:10X running buffer:50 mM NaOAc0.2M MOPS pH 7.010 mM EDTAfilter, store in dark at RT, pH to 7.0Formaldehyde gel:0.9% agarose in 1X running buffer, and 1ml formaldehyde per 20 ml gel. (For 200 ml gel, dissolve 1.8 g agarose in 170 ml water, let cool in 65 deg C water bath, add 10 ml formaldehyde (37% stock), and 20 ml 10X MOPS running buffer).1.5X Loading buffer:750 � formamide75 � formaldehyde (37%)150 � 10X running buffer15 � EtBr (5 mg/ml)10 � ddH20Best results if made fresh each timeLoading Dye:Bromophenol blue in 35% glycerol and 1X running buffer20X SSC:3M NaCl0.3M Na CitratepH 7.01. Pour gel in hood.2. Mix RNA (10�) with 1.5X loading buffer (e.g. 10 � RNA + 20� LB).3. Incubate 5 min at 65 deg C. Cool on ice.4. Add 2 � loading dye.5. Pre-run gel at 5 V/cm for 5 minutes. Load samples and run. Mix buffers after 3 hours.6. Wash gel 2X in water for 15 min, then 1X in 10X SSC for 15 minutes. Photograph gel under UV.7. Cut nylon membrane (MSI 0.45 �cron #N04HY00010) and several pieces of blotting (e.g. Schleicher and Schuell GB002) paper to the same size as the gel. Wet the nylon with dH20, then soak in 5x SSC.8. Assemble sandwich:a. Large sheet of plastic wrapb. Two pieces blot paper (precut to same size as gel) soaked in 20x SSCc. Gel (wells-side down)d. Presoaked nylone. One piece blot paper soaked in 5x SSCf. 10-15 pieces of dry blot paperh. Wrap whole sandwich in the plastic wrapi. Place glass plate and weight on top and let transfer >3 hr.9. Crosslink after blotting.10. Can probe either with non-radioactive probe or with radioactive probe .