TBE OLIGO Gel shift
OLIGO PREPARATION
Resolubilize oligo in 100ml of 10mM Tris pH 7.5/50mM NaCl
Measure OD 260/280nm and calculate concentration
Dilute 20mg into 200ml of 10mM Tris pH 7.5/50mM NaCl
Combine 400ml of complementary oligo in a new tube
Place in boiling H2O bath for 5 minutes and allow H2O to cool to room temperature
Recalculate concentration
LABELLING OLIGO
500ng annealed oligo (5ul)
5ml of 10X blunting buffer (!!Omit hot nucleotides from mixture!!)
3ml 32P dCTP (800Ci/mmol)
3ml 32P dGTP (800 Ci/mmol)
1.5ml Klenow (5U/ml)
H2O make volume up to 50ml
Incubate at RT for 30 minutes
Add 2ml of 5mM dNTPs
Incubate an additional 30 minutes
Pre run a G25 spin column (2 minutes @700 RCF)
Load the rxn onto G25 column
Spin down for 2 minutes @700 RCF
Discard properly of the column
Count 1ml and dilute a portion to 2x104 DPM/ml
TBE GEL RETARDATION ASSAY
Set up TBE gel with 0.25X TBE Running buffer in the cold room. Pre-run gel for 30 min
Mix 10X Ficoll binding buffer, .25-2mg (usually 1 mg) of poly dI:dC, 20,000 DPM of hot oligonucleotide, 1-5mg of protein extract and water to 15ml in a microcentrifuge tube. If desired add 5-100ng of cold oligo competitor for competition studies. Mix thoroughly and immediately place samples on ice for 20 minutes.
Add 1.5ml of 10X retardation gel loading buffer
Load gel and run at 210 V with 0.25 X TBE
Run until BPB is 2/3 way down gel
Transfer to Whatman paper and dry 30 minutes on gel dryer
Expose film for 4 hrs to O/N with screen @ -70 C
Reagents:
Acrylamide 40g/Bis-acrylamide 1.33g. Make volume up to 100ml and filter through .45mm filter
10X TBE
1M Tris base
1M Boric Acid
20mM EDTA tetrasodium salt
TBE RETARDATION GEL
| 25 ml 5% |
25 ml 4% |
50 ml 5% |
50 ml 4% |
|
|---|---|---|---|---|
| H2O | 20.6 ml | 21.2 ml | 41.2 ml | 42.4 ml |
| Acrylamide/Bis | 3.2 ml | 2.5 ml | 6.4 ml | 5 ml |
| 5 X TBE | 1.3 ml | 1.3 ml | 2.6 ml | 2.6 ml |
| 10% APS | 500ml | 500ml | 500ml | 500ml |
| Temed | 15 ml | 15 ml | 15 ml | 15 ml |
10 X Ficoll Binding buffer
50mM Tris pH 7.5
5mM EDTA
20% Ficoll
5mM DTT
375mM KCl
10X Blunting buffer (dG dC)
50ml 1M Tris pH 7.5
8ml 1M MgCl2
0.7ml B-Mercaptoethanol
2ml 100mM dATP
2ml 100 mM dTTP
37ml H2O
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