Nature research paper: Mechanism of mitoribosomal small subunit biogenesis and preinitiation
blasticidin S . Knockout cells were seeded in a 10-cm dish, 1 day before transfection, to achieve 70–90% confluency. Co-transfection of the expression plasmid pcDN5/FRT/TO and the Flp-recombinase plasmid pOG44 was carried out using Lipofectamine 3000 , according to the manufacturer’s instructions. Selective antibiotics, 100 μg mlblasticidin S were added 48 h post-transfection and culture media were replaced every 2–3 days.
Supernatant was collected and the pellet was resuspended in half of the volume of the MIBSM buffer used at the previous step and homogenized in teflon Dounce homogenizer. Homogenate was centrifuged 800for 15 min and supernatant was collected. The procedure with the pellet was repeated using half of the MIBSM buffer volume in the previous step. All supernatants were combined.
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