DNA Transfer 2. Denaturation: Wash gel in (0.5 M NaOH, 1.5 M NaCl) for 20' (small) to 30' (large). 3. Neutralization: Wash in (0.5 M Tris pH 7.0, 3.0 M NaCl) for 20' (small) to 30' (large). 4. Cut nylon membrane (MSI 0.45 micron #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. 5. Assemble sandwich: 6. Crosslink after blotting. OR 1. Set up a standard alkaline Southern transfer onto a nylon membrane with 0.4 M NaOH, 0.6 M NaCl (for positively charged membranes) or 0.25 M NaOH, 1.5 M NaCl (for uncharged membrane). Transfer >3 hours. 2. Crosslink if using an uncharged membrane. |
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