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.