FOOTPRINTING WITH DNASE1

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.