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(Jun-Ryul Huh)
pre-mix: 4.0µl dCTP 4.0µl dGTP 4.0µl dTTP 5.0µl 10x buffer 5.0µl primer/BSA 2.5µl [32P]dATP 2.0µl Klenow
Example: Human genomic DNA (Clonetech) 1) Mix DNA 100µl(10µg), NEBuffer2 40µl, HinIII(20u/µl) 2.5µl, incubate at 37°C for 2 - 2.5 hours 2) Add 800µl absolute EtOH to the tube, and invert for several times. 3) Vortex with highest speed for 2 min. 4) Spin at the highest speed for not longer than 5 min. 5) Remove the EtOH completely and dissolve with 25µl 2x BPB.
Denaturation and transfer solution: 0.5M NaOH + 1.5M NaCl Neutralizing solution: 0.5M Tris + 1.5M NaCl (pH7.4)
10µl labelled BAC probe 40µl cold nonspecific DNA 50µl poly dG-dT/dA-dC (0.25mg/ml) (Pharmacia) 200µl 1.5x SET (1x SET: 0.6M NaCl, 0,02M EDTA, 0.2M Tris·Cl [pH8.0], 2% SDS, 0.1% sodium pyrophosphate)