Fluorescently-labeled DNA (6FAM- or Alexa647-labeled on 3′ end,
purchased from IDT) was prepared by incubating complementary oligonucleotides
(Supplementary Table
3 ) at 95 °C for 5 min and slowly cooling down to room
temperature over ~2 h. For EMSAs, a 20 μl reaction containing 20
nM DNA was incubated with the indicated concentration of protein in the presence
of 50 mM HEPES pH 8.0, 150 mM NaCl and 1 mM TCEP. The reactions were incubated
for 30 min at 22 °C. After incubation, 5 μl were directly loaded
on a native polyacrylamide gel (6% DNA Retardation, Thermo Fisher) and run at 4
°C using 0.5 X TBE buffer for 60 min. The gel was then visualized using a
Typhoon Imaging System (GE Healthcare). Each binding experiment was repeated two
or three times (as indicated in figure legends) and
ImageJ44 (link) was used for quantification of percent DNA shifted (Fig. 4 ) or the mean intensity of free DNA and
standard deviation between the measurements (Extended Data Fig. 4c ).
purchased from IDT) was prepared by incubating complementary oligonucleotides
(
3
temperature over ~2 h. For EMSAs, a 20 μl reaction containing 20
nM DNA was incubated with the indicated concentration of protein in the presence
of 50 mM HEPES pH 8.0, 150 mM NaCl and 1 mM TCEP. The reactions were incubated
for 30 min at 22 °C. After incubation, 5 μl were directly loaded
on a native polyacrylamide gel (6% DNA Retardation, Thermo Fisher) and run at 4
°C using 0.5 X TBE buffer for 60 min. The gel was then visualized using a
Typhoon Imaging System (GE Healthcare). Each binding experiment was repeated two
or three times (as indicated in figure legends) and
ImageJ44 (link) was used for quantification of percent DNA shifted (
standard deviation between the measurements (