dsRNA formation and purification
dsRNA was formed and purified using the J1M1 RNAs described above. 40 µM
M1J1 dsRNA was formed in 1× FRET buffer by heating at 95°C for 5 min and
then slow cooling to room temperature. The reaction was stopped in 5×
stop/load buffer and separated on a pre-poured native gel (Novex 20%
TBE gel at 100 V for 1-3 h). The dsRNA was purified from the gel via
electrophoresis. Gel slices containing the dsRNA were placed in
preparative well of a 1% agarose gel. Four to six 1-inch pieces of
Whatman paper were placed in the prep well (between the gel slices and
the gel) the dsRNA was electrophoresed (100 V for 3 min) from the gel
slices onto the Whatman paper. The Whatman paper was imaged on a BioRad
ChemiDoc Gel Imager to verify dsRNA migration. Each Whatman paper was
placed in a 1.7-ml light sensitive Eppendorf tube with 500 µl of 10×
MgCl2 FRET buffer
(500 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM NaCl, 10
mM DTT). Tubes were placed in a microshaker (1400 rpm, 4°C) overnight to
extract dsRNA. The 10× MgCl2 FRET buffer
(~500 µl) was removed from the Eppendorf tubes and added
to 3 volumes of ethanol (EtOH), 0.3 M sodium acetate, and 15 µg of
GlycoBlue (Invitrogen). RNA was incubated at -20°C overnight. RNA was
precipitated at 19000 × g for 45 min at 4°C. Pellets were washed
with 200 µl of 70% EtOH and spun again at 19000 × g for 20 min
at 4°C. The supernatant was decanted and RNA was resuspended in water.
The purified dsRNA was quantified by nanodrop and used for the strand
displacement gel assay.