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.