At selected time intervals, buffer solution was taken for UV spectrometer and replaced with fresh buffer solution.
Protein-DNA-bead complexes were washed 2 times in lysis buffer (no inhibitors), 2 times in lysis buffer containing NaCl (360 mM), 2 times in wash buffer and 1 time in TE buffer, for 15 min in each individual solution.
Cells were pelleted by centrifugation 6000 rpm at 8 min and then washed 2 3 times with certain buffer for each strain.
The blots were washed three times with TNTT buffer for 5 min each.
Larvae were washed three times in staining buffer for 10 min at room temperature.
Worms were washed 3 times in cacodylate buffer for 10 min each wash, followed by a secondary fixation in 1% osmium tetroxide for 2 hours at room temperature.
Precipitated total DNA was dissolved in 50 µl 10 mM Tris/HCl (pH 8.0) and diluted 500 times in this buffer for most PCR reactions.
Following this incubation, membranes were washed three times using HYB100 buffer for 10 min, wrapped in Saran wrap, and exposed to film overnight.
Cells were suspended in 100 uL of fixative (5% paraformaldehyde in wash buffer), incubated at room temperature for 90 minutes then washed three times with wash buffer for five minutes.
The streptavidin-protein complexes were boiled in 2 times SDS sample buffer for 5 minutes (BioRad), resolved by SDS-PAGE, transferred to a PVDF membrane, and blocked in Tris buffered saline (TBS) containing 5% milk.
Slides were then washed seven times in TNT buffer for 3 minutes per wash, once in PBS containing 1% Triton X to minimize background fluorescence (3 minutes) and a final wash in TNT buffer (3 minutes).
