DNaseI footprinting
This method works well for binding conditions in which close to 100 % binding is
achieved (assayed by gelshift experiments). The amounts of DNaseI and incubation time
may vary.
* prepare DNA-fragment (100 to 300 bp in length) by cutting it with one 5' overhangproducing restriction enzyme and one 3' overhang-producing restriction enzyme
* label DNA-fragment at the 3' end using a Klenow-DNA Pol. fill-up reaction or a
SuperScript RT reaction
* setup binding reaction in 20 µl total volume:
10000 - 20000 cpm labeled fragment, 4 µl 5 x DNaseI binding buffer, protein, H2O
* incubate 10 min at room temperature
* dilute DNaseI ( i.e. RNase free DNaseI 10 U/µl; Roche Diagnostics)
with DNaseI dilution buffer to 0.5 - 1 U/µl
* add 1 to 3 U DNaseI to each binding reaction
* incubate 60 to 120 sec. at room temperature
* stop reaction by adding 100 µl phenol, vortex immediately
* add 80 µl H2O, 100 µl chloroform, vortex
* spin for 2 min in tabletop centrifuge
* disgard supernatant, add 3 µl glycogen (2 µg/µl), 15 µl 3 M NaOAc, 600 µl Ethanol
* incubate at least 30 min. at -80 °C
* spin 10 min. in tabletop centrifuge, wash with 70 % Ethanol, dry pellet
* resuspend pellet in 3.5 µl sequencing stop solution
* heat sample 3 min. at 95 °C, chill on ice
* load onto 8 % sequencing gel
* as marker we use pBR322 MspI cut, labeled with T4 Polynucleotide kinase
Buffers:
5 x DNaseI binding buffer: (recipe may vary for different proteins, this is just one
example)
100 mM Tris pH 7.5; 500 mM NaCl; 25 mM MgCl2; 5 mM EDTA; 25 % glycerol; 0.1
µg/µl poly[dIdC].
DNaseI dilution buffer:
2 mM MgCl2; 2 mM CaCl2; 20 mM Tris pH 7.5; 25 % glycerol.
sequencing stop solution:
95 % Formamide; 20 mM EDTA; 0.05 % Bromphenol blue and Xylene cyanole FF each.