| John Mundy, Institute of Molecular Biology, Copenhagen, Denmark http://www.arabidopsis.org/info/Protocols_Mundy2.jsp#Footprint 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
1ul probe (0.1©0.5ng or 10©20kcpm)
0.15ul 20mM EDTA
0.4ul 10ug/ul dIdC or dAdT (from gel shift assay)
0.5ul 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 ©20C.
Try 3 different [s] ofDNAse mix (A,B,C)
1,2 & 3ul stock DNAse1
2 ul 1M MgCl2
©> 100ul H2O
4)binding rxn: components titrated & optimized by gel shiftassays
2ul probe mix
Xul extract
©>18ul NEB (see nucprp.ptc)
30' RT
5)DNAse rxn: add 2ul DNAse mix to binding rxn
inc 1' RT
stop w/ 100ul DNAse stop mix:
stock/50ml
6M Urea 18g
0.4M NaCl 6.6ml 3M
1% SDS 5ml 10%
20mM EDTA 4ml 250mM
10mM Tris 8 0.5ml 1M
0.8M NH4OAc 5ml 8M
10ug/ml glycogen 50ul 10mg/ml
5)P/CHCl3 ext
6)EtOH ppt
7)PAGE: Resuspend carefully in 8ul sequencing sample buffer (5'vortex, 5' 60C, 1'
vortex, 2' 90C, spin, transfer to new tube,count cpm). Load equal counts on 6% or gradient sequencing gel.
Notes: If extract inhibits DNAse, add 0.1©0.3ul extra DNAse mixto binding rxns.
DNAse requires Mg, some factors are inactivatedby it! Remember ug/KB x 0.66 = picomole thus 1ng of 300bp probe =2 femptomole.
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