Briefly, 1 ul of genomic DNA (diluted to 10 ng/ul) was added to 9 ul sample buffer and was heated to 95 °C for 3 min to denature the template DNA.
Beads were washed with RIPA buffer for three times and were heated in SDS loading buffer.
In separate microcentrifuge tubes, 50 ml of each Saccharomonospora extract and gold nanoparticle reaction supernatant was mixed with equal volumes of ×2 Laemmli sample loading buffer with 2-mercaptoethanol and were heated at 95°C for 5 min, and 20 μl of each of the samples was resolved on a tris-glycine (4% to 20%) minigel.
Purified mRNA was fragmented by addition of 5× fragmentation buffer (Illumina, Hayward, CA) and was heated for 5 min at 94°C in a thermocycler.
The purified mRNA was fragmented by addition of 5x fragmentation buffer (Illumina, Hayward, CA) and was heated at 94°C in a thermocycler for 8 minutes.
Purified mRNA was fragmented by addition of 5X fragmentation buffer (Illumina, Hayward, CA) and was heated for 5 min at 94° C in a thermocycler.
The purified mRNA was fragmented by addition of 5× fragmentation buffer (Illumina, Hayward, CA) and was heated at 94°C in a thermocycler with 2 different times (2 min and 5 min).
Immunoprecipitates were washed 3 times in high salt buffer, and beads were heated to 100°C in a buffer containing 25 mM Tris pH 7.6, 250 mM NaCl, 0.5% SDS and 5 mM EDTA for 5 min. Half of the mixture was heated with 6×Laemmli sample buffer, resolved in an SDS-polyacrylamide gel, transferred to nitrocellulose and probed with antibody to CDK9.
After washing (3 times with high salt (500 mM NaCl) and twice with isotonic buffer), beads were heated with SDS-PAGE sample buffer for 10 minutes at 70°C and proteins were resolved by 4 12% gradient SDS-PAGE.
