FOOTPRINTING WITH DNASE1

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 [s] 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.