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) 0.15μl 20mM EDTA 0.4μl 10μg/μl dIdC or dAdT (from gel shift assay) 0.5μl H2O
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 ofDNAse mix (A,B,C) 1,2 & 3μl stock DNAse1 2 μl 1M MgCl2 ©> 100μl H2O
4)binding rxn: components titrated & optimized by gel shiftassays
2μl probe mix Xμl extract ©>18μl NEB (see nucprp.ptc) 30' RT
5)DNAse rxn: add 2μl DNAse mix to binding rxn
inc 1' RT stop w/ 100μl DNAse stop mix:
stock/50ml6M Urea 18g0.4M NaCl 6.6ml 3M1% SDS 5ml 10%20mM EDTA 4ml 250mM10mM Tris 8 0.5ml 1M0.8M NH4OAc 5ml 8M10μg/ml glycogen 50μl 10mg/ml
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.
