UV-Crosslinking
A) Design Primers and Oligos
1.) Order oligos (sense and antisense) with at least 10 basepairs overhang on each side of bindingside.
Order primers at least 10 basepairs long flanking bindingside. (See example of Mef-3 or Cef-1 in folder).
2.) Resuspend oligos and primers (lyophilised) in 100 ul of H2O each. Take O.D. Dilute oligos to 100 ug/ml and primers to 4x molar concentration of oligos.
B.) Annealing and labeling reaction
1.) Set up annealing reaction: oligo (sense/antisense) 100ng/ul 1ul
primer (antisense/sense) 4x MW 1ul
H2O 7ul
10x low salt buffer 2ul
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11ul2.) Incubate 5min. at 65 C, 10min. at RT, 15min. on ice.
3.) Then add: dATP (25mM) 2ul (Pharmacia cat. 27-2050-01)
dGTP (25mM) 2ul ( " cat. 27-2070-01)
Brd-UTP(25mM) 2ul (Sigma cat. B-0631)
dCTP* (800 Ci/mmol) 2ul (Amersham cat. PB 10385)
Klenow 1ul
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20ul4.) Incubate 30min. at RT, 10min. at 68 C.
5.) add 2ul DNA-LB directly and purify on 10% acrylamide TBE gel. (use labeling protocol).
6.) In am resuspend oligos in 10mM Tris/50mM NaCL to about 100000 cpm/ul.
C.) Gel-Shift-Reaction
1.) In am run TG/TBE gel-shift (usually 5% acrylamide) per protocol. Use 1.5 mm spacer.
2.) Scale up binding reaction 3x - 10x according to preexperiments. Do not dry gel.
3.) After run separate plates, cover with saranwrap and crosslink gel in UV-transilluminator at 2500 uJoule. Then wetexpose gel O/N in wet cassette (Sigma cat. E-8635,see protocol methylation interference for details).
4.) In am cut out band of interest, unspecific band and free counts. Electroelute (see protocol).
D.) Electroelution and Proteinelectrophoresis
1.) After the elution recover the counts and spin down to 100ul at 6000 rpm , for 45min.in Beckmann-centrifuge, rotor J-17. (Do not leave O/N). Then backspin same speed for 20min additional samples. Pool in one column. Concentrate again.
2.) Add 1ml of 1x 0.1% SDS-sample buffer (pH 6.8 to adjust pH of sample) to column and spin 30min, same speed. Repeat 1x , then repeat until protein is concentrated to 100ul. Then backspin 20min., same speed, to recover protein-DNA-complex.
3.) Add 10% SDS-solution to sample to a final of 1% SDS.
4.) In meantime pour 7%-10% SDS-gel , 1.5 mm spacer per standartprotocol (red-book).
4% stacking gel.5.) Add 1ul/100ul sample of beta-ME and 1ul/100ul of regular DNA-LB.
6.) Boil sample 3min., as well radioactive marker (20-25 ul).
Load equal amount of counts, otherwise multiples of eachother. Run O/N at 5-10 mA. (10mA run for 12 hours in 9% gel). For shorter run use 20mA through stacking and 40mA through separating gel.
7.) Dry gel for 1 hour and expose O/N.
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