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1) Run gel (~50V for 6 hrs) in 1XTBE buffer 2) Soak gel in 0-25M HCl for 10 minutes. 3) Rinse gel briefly in H2O. 4) Prewet Nylon membrane in H2O (Biotrans + of ICN). 5) Transfer gel using 0.4M NaOH as the transfer buffer. (O/N.) 6) Rinse membrane for 5 minutes in 2X SSC. 7) Probe or can be stored in -20°C for a long period of time. Prehyb: 50µg/ml tRNA + 40% deionized formamide (moderate stringency; high=50%, low=30%) + 50% 2X Denhardt's Solution + H2O to 10%. Prehyb at any temperature for 4 hours. Add Probe: Hybridize at 50°C (formamide buffers temperature to be between 40-50°C.) Washing: At 48°C 1X 15 minutes in 2X SSC, 0.2%SDS 2X 15 minutes in 1X SSC, 0.2%SDS Expose to phosphoimager screen, then to x-ray film. |