1)probe: same as used for gelshift), isolated by isotacelectrophoresis w/out EtOH ppt which can enature dsDNA 2)probe mix/rxn: volumes x # samples 1μl probe (0.1©0.5ng or 10©20kcpm) 3)DNAse mix: made up near end of binding incubations. DNAse l(Worthington DPFF,Cat#LS0006330, lot #58A047,5mg) is 1mg/ml in150mM NaCl, 50% glycerol, store at ©20℃. Try 3 different [s] ofDNAse mix (A,B,C) 4)binding rxn: components titrated & optimized by gel shiftassays 2μl probe mix 5)DNAse rxn: add 2μl DNAse mix to binding rxn inc 1' RT stock/50ml 5)P/CHCl3 ext 6)EtOH ppt 7)PAGE: Resuspend carefully in 8μl sequencing sample buffer (5'vortex, 5' 60℃, 1' vortex, 2' 90℃, spin, transfer to new tube,count cpm). Load equal counts on 6% or gradient sequencing gel. Notes: If extract inhibits DNAse, add 0.1©0.3μl extra DNAse mixto binding rxns. DNAse requires Mg, some factors are inactivatedby it! Remember μg/KB x 0.66 = picomole thus 1ng of 300bp probe =2 femptomole. |
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