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1% Formaldehyde Gel: 1.52 g agarose 113 mL H2O Microwave to melt add 15.2 mL 10 x MOPS Buffer 24.65 mL Formaldehyde NO EtBr Total volume 152 mL Running buffer: 1 x MOPS Buffer (not DEPC-treated, no formaldehyde) Sample Preparation: a) Add up to 15µL of RNA (1-5µg ploy(A) or 5-50µg total RNA) to a microfuge tube b)Add 16µl of the Sample Buffer Stock Solution to each RNA sample; heat to 65°C, 10', cool on ice, add 4µL RNA dye solution, spin, load the agarose gel. NOTE: the EtBr in the sample buffer will bind irreversibly to the RNA during the heating to 65°C. The unbound EtBr will migrate up the gel while the RNA-bound EtBr will run down the gel without affecting the mobility of the RNA. The RNA can be visualized while running and no post-electrophoresis staining is required for photography. (see Rosen and Villa-Komaroff BRL Focus 12, #1, pg. 23-24) Running the gel: 50 vols (constant voltage) Post electrophoresis processing: a) The gel is photographed while still on the gel tray. Minimize the exposure to the UV light while taking the photograph. b) Transfer the gel to a large baking dish containing dH2O c) Change the dH2O after 2 min. d) Pour off the dH2O after 5 min. and add enough 50mM NaOH to cover the gel well. e) Incubate with gentle shaking for 30 minutes. f) Pour off the naOH, add an excess of 100mM Tris HCl pH 7.0 g) Make 3 changes of this buffer, 10 min. each. h) Finally soak the gel in 10 x SSC for 5 min. i) Set up capillary blotting apparatus with layers of: 1) Whatman 3MM in contact with the transfer buffer (10 x SSC) 2) The Gel 3) The blotting membrane (Zetaprobe, BioRad) 4) Whatman 3MM 5) Lots of paper towels NOTE: Make sure that there are no bubbles under the gel, under the blotting membrane or on top of the blotting membrane. j) Transfer overnight with copious 10 x SSC and several changes of paper towels. Processing the Blot, Prehyde, and Hybridization: a) Disassemble the blot and transfer the blotting membrane to a tray of 2 x SSC. b) Wipe lightly with a Kimwipe and a gloved hand to remove any adhering agarose. c) UV crosslink on a Stratalinker on Autocrosslink. d) Transfer back to a tray of 2 x SSC e) Place the blot into a Seal-a-meal bag and add the Prehybe solution (~30 ml for a small blot ~50-75 ml for a large blot. This is a GREAT EXCESS of Prehybe solution and you can probably get away with ~5 ml for a small blot and ~10ml for a large one. I normally make 30-50 ml of Prehybe solution and reserve a small amount for the Hybridization solution. The remainder I add to the blot for the Prehybe.) f) Prehybe at 65°C for >1hr g) Remove the Prehybe solution by cutting a hole in the corner of the Seal-a-meal bag and using a 25ml pipette like a rollng pin to squegee out the Prehybe. h) Add the 32P-labeled probe (after heating to 100°C for 5 min to denature) an aliquout of the Prehybe Solution which had been reserved prior to the prehybridization. i) Transfer this olution to the Seal-a-meal bag containing the blot. Seal the bag and place it inside a second bag. j) Incubate at 65°C overnight. k) Transfer the Blot to a tray containing Wash Buffer 1 at 65°C. Wash with three changes, 15 minutes each. l) Wash with three changes of Wash Buffer 2 at 65°C, 15 minutes, each. m) Wash with one change in 40 mM PB Buffer at room temperature. n) Wrap in Saran Wrap and expose overnight at -80°C with screens. SOLUTIONS 10 x MOPS Buffer 0.2 M MOPS (Na salt) 0.05 M Na Acetate 0.01 M Na2EDTA adjust to pH 7.0 store in a dark bottle or foil wrapped bottle Sample Buffer Stock Solution: 5µl 10mg/ml EtBr 250µl Formamide 50µl 10 x MOPS Buffer 80µl Formaldehyde RNA Dye Solution 40% sucrose 0.025% Bromophenol blue (6mg/25ml) Prehybe Solution 75 ml PB buffer 0.3 ml 0.5 M EDTA 52.5 ml 20% SDS 1.5g BSA (good quality BSA) 1.5 ml 10mg/ml denatured salmon sperm DNA 20.7 ml H2O Hybe Solution Prehybe solution + Denatured probe PB Buffer 67g Na2HPO4-7H2O ~2 ml 85% H3PO4 pH 7.0 H2O to 500 ml, autoclave Wash Buffer 1 (used at 65°C) 40mM PB Buffer 1% SDS 1mM EDTA 0.5% BSA (fraction V, junk grade) Wash Buffer 2 (used at 65 °C) Wash Buffer 1 except no BSA |