6 Matching Annotations
  1. Jun 2019
  2. May 2019
    1. DNA sequencing
    2. Automated DNA sequencing on plasmid templates or on PCR products was carried out with dye terminator cycle sequencing kits from Perkin-Elmer on an automated sequencer (model 377, Applied Biosystems), following the manufacturer’s instructions
    1. GST fusions of yeast RNA Pol I subunits were purified as described in (Werneret al., 2010). Yeaststrainsover expressing GST tagged RNA Pol I proteins were grown overnight at 30°C in 10 mL of SC-Ura medium with 2% glucose medium. Cells were pelleted, washed in SC-Ura with galactose. Protein expression was induced by transferring the entire pellet into200 mL of SC-Ura with 2% galatose to give a final OD600of 0.8-1.0. For proteins A190 and A43 that express at very low levels, the overnight culture volume and induction volume were doubled. Cells were cultured at 30°C harvested at 3.0-5.0 OD and washedwith ice cold water. The cell pellet was suspended in 5 mL of ice cold Buffer A (Section, 750 μL ofcell suspensions were aliquoted into 1.5 mL microfuge tubes and to this 500 μL glass beads were added. Cells were lysed by bead beating using a vortex mixer (VortexGenei -2 with mix-mate attachment), and the lysate was centrifuged at high speed for 15 min at 4°C. Supernatants were dispensed into a 15 mL conical tube and Triton X-100 was added to a final concentration of 1%. Pre-swollen glutathionebeads were washed in Buffer B(Section which 200 μL of 1:1 bead suspension was added to approximately 5 mL of A34 and A43 expressing cell lysate and 100 μL of 1:1 bead suspension was added to 5 mL of A190 cell lysate and incubated for 2 h at 4°C on a rotary mixer. Lysates were centrifuged at 5000 xgfor 2 min and the beads were washed with ice cold Buffer C (Section twice. Beads were further washed with ice cold Buffer B followed by ice cold 1X phosphate buffered saline (PBS) twice. Beads were suspended in an equal volume of 1X PBS with protease inhibitor cocktail (Sigma)
    2. Protein expression and purification from yeast