Problem (Step 14): The bands are faint or absent. Transfer and while the membrane is still wet (Step 6.ii), “wash” the side of the membrane in contact with the gel in 1× electrophoretic Polyacrylamide sticking to the membrane can cause high background by retaining the excess probe or unincorporated label. When preparing the prehybridization/hybridization mix, include tRNA and/or poly(A) at a concentration of 200 µg/mL.ģ. Try prehybridizing for a longer period of time (Step 8).Ģ. Solutions: High background may be reduced by one of the following approaches.ġ. Problem (Step 14): A high background is observed on the membrane. Previous Section Next Section TROUBLESHOOTING Replace the buffer with an appropriate volume of fresh prehybridization/hybridization mix containing radiolabeled probe atġ0 6 cpm/mL, and hybridize overnight at 42☌. Prehybridize the membrane with shaking orĩ. Mix, using 10 mL for a heat-seal bag or ∼3–5 mL for a small hybridization tube. Place the membrane in a heat-seal bag or cylindrical glass hybridization bottle and cover with prehybridization/hybridization Surface or bake the membrane in a vacuum oven for 1 h at 80☌.įor microRNAs (miRNAs RNAs <50 nucleotides), covalent chemical cross-linking ( Pall and Hamilton 2008) may be used to fix the small RNAs to the nylon membrane.Ĩ. To cross-link the RNA to the nylon transfer membrane, place the membrane in a Stratalinker with the RNA surface facing up.Įxpose it to short-wave UV light (254 nm the 1200 mJ auto-cross-linking setting in a Stratalinker) for 1 min.Īlternatively, use a 254-nm UV transilluminator, exposing the membrane with the RNA surface facing down against the glass Wrap the membrane in plastic wrap and place it on a sheet of Whatman 3MM paper.ħ. Carefully remove any gel fragments from the membrane and mark the orientation of the membrane with a ballpoint ink pen. Carry out the transfer at 250 mA for 2 h at 4☌ and then at 350 mA for a further 2 h at 4☌.Ĭross-Linking, Prehybridization, and HybridizationĦ. Place the assembled gel cassette into the electroblot chamber and fill the chamber with 1× electrophoretic transfer buffer, The RNA will migrate to the positive (+) pole.ĥ. Nylon membrane, two sheets of Whatman 3MM paper, sponge. The layers of the sandwich are as follows, starting with the negative (−) pole: sponge, two sheets of Whatman 3MM paper, gel, Add the second sponge on top of the Whatman paper sheets and clamp the cassette closed. Wet two more sheets of Whatman 3MM paper with 20× SSC, and place them on top of the membrane. Place the nylon membrane on top of the gel and remove any bubbles. Float the nylon transfer membrane in H 2O until wet, then immerse it in 20× SSC. Transfer the gel with the paper onto the sponge, such that the paper is on top of the sponge. Soak a sponge from the electroblot apparatus in electrophoretic transfer buffer until wet. Wet two sheets of Whatman 3MM paper with 20× SSC and place them on top Remove the gel from the tank and separate the plates. For details, see Polyacrylamide Gel Electrophoresis of RNA ( Rio et al. Run the gelīetween 15 and 20 W on constant power, usually for 3–4 h, or until the bromophenol blue (in the formamide gel-loading buffer) Load the samples and the RNA marker ladder onto a high percentage (10%–15%) denaturing polyacrylamide–urea gel. Resuspend each RNA sample (∼10–15 µg of total RNA) in 8–12 µL of formamide gel-loading buffer. Whatman 3MM chromatography paper (four sheets must be the same size as the gel see Steps 3 and 4)ĭenaturing Polyacrylamide–Urea Gel and Electrophoretic Transferġ. Stratalinker (Stratagene) with 254-nm bulbsĪlternatively, use a 254-nm UV transilluminator or vacuum oven set at 80☌ (see Step 7). The gel is typically ∼18 × 18-cm, 1.5 mm thick, set with a 20-well comb. Polyacrylamide gel electrophoresis system and power supply Oven with shaker or hybridization oven with a rotating rack for glass hybridization bottles, set at 42☌ The membrane should be cut to the same size as the gel. Nylon transfer membrane (positively charged e.g., GE Healthcare, Hybond-N+) If heat-seal bags are used, a heat sealer (e.g., National Instruments, Model 310) will be required.Įlectroblot transfer tank and power supply (e.g., Bio-Rad Trans-Blot cell with wire electrodes and PowerPac HC Power Supply) Autoradiography or phosphorimaging equipmentĬontainers for hybridization (polyethylene heat-seal bags or cylindrical glass hybridization bottles)
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