TGE OLIGO Gel shifts
Michael S. Parmacek
Reagents:
1) Acrylamide/bis-40g acrylamide/1.33 g bisacrylamide make volume up to 100 ml and filter thru .45uM filter
2) 5X Tris-Glycine Running Buffer:
BME 35 ul
Tris Base 60.56 g
Glycine 285.4 g
EDTA (tetra sodium) 7.60 g
H2O make volume up to 2L
3) 10 % Oligo labelling/purification gel:
acrylamide/bis 12.5 ml
10X TBE 5 ml
10% APS 250 ul
H2O 32.5 ml
temed 50 ul
4) 10X TGE Binding Buffer (50 mM NaCl):
(NOTE: can also use 100 mM NaCl, 25 mM NaCl and 40 mM KCl Binding Buffers):
5M NaCl 100 ul
1M Tris 7.5 100 ul
500 mM EDTA 20 ul
1M DTT 50 ul
100 % glycerol 400 ul
BSA (10 mg/ml) 100 ul
H2O 220 ul
5) TGE Retardation Gels (.75 mM thick, 14 well comb):
4% 5%
H2O 17 ml 17.5 ml
Acrylamide/bis 3.1 ml 2.5 ml
5X TGE 5 ml 5 ml
10% APS 500 ul 500 ul
temed 15 ul 15 ul
6) Poly dI:dC (Pharmacia)
7) TBE Retardation Gels:
.25X TBE/5%
H2O 20.6 ml
Acrylamide/bis 3.2 ml
5X TBE 1.3 ml
10% APS 500 ul
temed 15 ul
OLIGO PREPARATION
Resolubilize oligo in 100 ul of 10 mM Tris 7.5/50 mM NaCl.
Measure E 260/280 and calculate concentration.
Dilute 20 ug into 200 ul total of 10 mM Tris/50 mM NaCl.
Combine 400 ul of complementary oligo in a new tube
Place in boiling H2O bath for 5 minutes and allow H2O to cool to room temperature.
Recalcuate concentration.
LABELLING OLIGO
500 ng annealed oligo
5 ul of 10 X blunting buffer (omit hot nucleotides from mixture)
3 ul 32P dCTP (800 Ci/mmol)
3 ul 32P dGTP (800 Ci/mmol)
1.5 ul Klenow (5 u/ul)
H2O make volume up to 50 ul
Incubate at RT for 30 minutes
Add 2 ul of 5 mM dXTPs
Incubate an additional 30 minutes
Add 5 ul of 10X DNA loading Buffer
Load onto 10% Oligo labelling/purification gel run at 300 V
Run until the BPB is 1/2 way down.
Separate plates and cover gel with Saran wrap.
Expose film wet for 5-10 seconds.
Place film under plate with gel and align to locate labelled oligonucleotide.
Cut out band with a razor band and elute over nite in 500 ul of 0.5 M NH4AC/5mM EDTA at 37°C with shaking.
In AM remove eluate, add 2 volumes of 100% ETOH and precipitate on dry ice.
Dry and count dry pellet.
Resolubilize in 500 ul 10 mM Tris/50 mM NaCl
Count 1 ul and dilute a portion to 2x104 DPM/ul
TGE GEL RETARDATION
Mix 10X binding buffer, 250 ng-2ug poly dI:dC, 20,000 DPM of hot oligo, 1-5 ug of protein extract, and water to 15 ul in a microfuge tube. If desired add 5-50 ng of cold oligo competitor for competition studies.
Dilute poly dI:dC in 10 mM Tris/50 mM NaCl
Incubate at room temperature for 15 minutes.
Add 1.5 ul of 10X retardation gel loading buffer (.25% bromphenol blue/10 mM Tris)
Load gel and run at 125 V with 1X TGE Running Buffer
Run BPB 2/3 way down gel
Transfer gel to Whatman and dry 30 minutes on gel dryer.
Expose film for 4 hrs to o/n with screen at -70°C
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