GELSHIFT

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 3μg/μl

2)Probe preparation: see endlabl.ptc & isotach.ptc. probe shouldbe 10©20k cpm/μl.

Fragments larger than 400bp should not be used. Make A stock (25μg/50μl) of probe plasmid digested at one end w/ 5' overhang.

3)Binding rxn:

1-2μl probe (0.5ng or 20k cpm in isotach 40mM Tris 7.5 buffer)

1-2μl poly dIdC or dAdT (3μg/μl in NEB, sonicated to 300©500bp)

1-6μl extract (5©10μg/μl in NEB)

>10μl NEB (see nucext.ptc)

incubate 30' RT

1μl sequencing dyes immediately prior to loading under tension

4)Titrations: Start w/ extract titration at 3μg/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

50μl TEMED

400 μl 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 60℃

60μl TEMED

100μl 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 80℃ on dryer.Expose o/n -70℃ w/screen.