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Labeling Oligo's for Gel Shift
Must Have 51 Overhang & Have at Least 1 'IC" to Fill-in
oligo (2 ug) 1OX Hind Buf.
10 X hind buf. 2 ul .5 M Kaol
10 mM d ATP 1 ul 60 mM Tris 7.5
10 mM d GTP 1 ul 60 mM MgCl2
10 mM d TTP 1 ul 10 mM DTT
32 P-d CTP 10 ul
10 mM DTT 2 ul
H20
20 ul
Klenow 4 ul
451 at 370
50 mM Tris ph 7.5
add 180 ul Col. Buf. 100 mM Nacl
0.2% SDS
-put over spin col.
EtoH ppt
resus. in loo ul 10 mM Tris pH 7.5
50 mM Nacl
To make DS oligo's for this procedure put 51 overhang restriction site
Anneal complimentary SS oligo's in 10 mM Tris 7.5, 50 zaM Nacl
Heat to 750 X 10 Cool slowly to RT, then ice
Spin Col
-1 ml syringe
-pack with small amount of glass wool -G-50 Sephadex (course) -spin to pack - 511 at 2K -add labeled mix -spin 151 @ 2K
GEL Shift
GEL (1 GEL)
30% Acrylamide 6.7 ml
2% Bis 5 ml
10 X TBE 1.25 ml (.25 X TBE)
H20 36.75 ml
10% APS 0.5 ml
TEMED 15 ul
Running buffer-(.25 X TBE)
pre-run gel - 2 hours until mamps drop to 10 (starts @ 25 mamps,
isov)
run gel 15OV, 10 mamps
mix 10 X GEL Shif Buf.
Extract 100 mM Tris pH 7.5
probe 20 ng 400 mM Nacl
-comparator 200 ng 10 mM EDTA
(1 mg/ml) pdI/dc (pharmacia) 10 mM DTT
10 X gel shift buffer
H20
25 ul total - I do these reactions in cheap (non sterile) 96 well plates
201 @ 25- (R.T.)
add 2.5 ul dye (BPB/sucrose)
lox
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