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 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)DNA se mix: made up near end of binding incubations.DNA se l(Worthington DPFF,Cat #LS0006330,lot #58A047,5mg)is 1mg/ml in150mM NaCl,50% glycerol,store at ©20℃. Try 3 different [s] ofDNA se mix (A,B,C) 1,2 & 3μl stock DNA se1 2μl 1M MgCl2 ©> 100μl H2O 4)binding rxn: components titrated & optimized by gel shiftassays 2μl probe mix Xul extract ©>18μl NEB (see nucprp.ptc) 30' RT 5)DNA se rxn: add 2μl DNA se mix to binding rxn inc 1' RT stop w/ 100μl DNA se 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 10μg/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 DNA se,add 0.1©0.3μl extra DNA se mixto binding rxns. DNA se 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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