John Mundy, Institute of Molecular Biology, Copenhagen, Denmark
http://www.arabidopsis.org/info/Protocols_Mundy2.jsp#Gelshift 1)Nuclear extract: see nucprep.ptc & nucext.ptc extracts should be at least 3ug/ul
Probe preparation: see endlabl.ptc & isotach.ptc. probe shouldbe 10©20k cpm/ul.
Fragments larger than 400bp should not be used. Make A stock (25ug/50ul) of probe plasmid digested at one end w/ 5' overhang.
3)Binding rxn:
1-2ul probe (0.5ng or 20k cpm in isotach 40mM Tris 7.5 buffer)
1-2ul poly dIdC or dAdT (3ug/ul in NEB, sonicated to 300©500bp)
1-6ul extract (5©10ug/ul in NEB)
>10ul NEB (see nucext.ptc)
incubate 30' RT
1ul sequencing dyes immediately prior to loading under tension
4)Titrations: Start w/ extract titration at 3ug/rxn poly dIdC.
At extract [] w/ max binding, do dIdC titration.
work for complete probe binding, none free. Try Mg salts later.
5)Competitions: Fragments should be isolated by PAGE/isotach. 10-100ng DNA/rxn is usually necessary.
6)gels:
Acryl
48.5ml
10ml 30/0.8% acryl stock
1.5ml 10x TBE
50ul TEMED
400 ul 10% APS
run 100©200v w/ circulation
Acryl/agarose gel
H2O80ml H2O
0.7g agarose
boil to dissolve
10ml 10x buffer 100mM Tris 7.5, 10mM EDTA, 30mM NaOAc
10ml 30/0.8% acryl stock
cool to 60C
60ul TEMED
100ul 10% APS
let set for 2hr, prerun with circulation 30' at 100v and run w/ circulation
7)Gels are dried unfixed on Whatman DE 81 sheets at 80C on dryer.Expose o/n -70C w/screen.
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