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Net367250uc

Manufactured by PerkinElmer
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The NET367250UC is a laboratory equipment product manufactured by PerkinElmer. It serves as a core function for laboratory applications. A detailed description while maintaining an unbiased and factual approach is not available.

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Lab products found in correlation

Nanodrop one onec uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions) Rneasy rna clean up kit, by Qiagen (1 mentions) Allprep dna rna mini kit, by Qiagen (1 mentions)

2 protocols using net367250uc

1

Pre-rRNA Pulse Chase Assay Protocol

Cited 1 time
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Pre-rRNA pulse chase assays were performed as previously described62 (link), with minor modifications. Briefly, 50 mL of cells were grown at 30 °C in EMM-ura to OD600nm of ∼0.5–0.8. The cells were then diluted to an OD600nm of 0.018 (WT) or 0.03 (Pnmt1-seb1) where 60 µM of thiamine was added for 15 h at 30 °C. Cells were resuspended in 850 μl of EMM-ura containing 125μCi of uridine-3H (Perkin Elmer, cat no: NET367250UC). Following a 4 min pulse, 200 μl of cells were diluted in 1.7 mL of EMM supplemented with uracil and 60 μM of thiamine. At various times point, cells were taken and rapidly frozen in liquid nitrogen. RNA was purified and separated as described in the ‘preparation and analysis of RNA’ section, with the following modifications: 10,000 cpm of radioactivity from each sample were loaded and migrated onto a 0.8% agarose-formaldehyde gel. Finally, a tritium screen (BAS-IP TR 2025 E Tritium Screen, 28956482, Cytiva) was added to the membrane and analyzed suing a typhoon trio instrument.
Duval M., Yague-Sanz C., Turowski T.W., Petfalski E., Tollervey D, & Bachand F. (2023). The conserved RNA-binding protein Seb1 promotes cotranscriptional ribosomal RNA processing by controlling RNA polymerase I progression. Nature Communications, 14, 3013.
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2

Quantitative Analysis of rRNA Synthesis

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Cells were labeled with 1 μCi/mL 5,6-3H-uridine (Perkin Elmer #NET367250UC) or 2,8-3H-adenine (Perkin Elmer #NET063001MC) for times indicated in the figure legends. Total RNA was extracted using the AllPrep RNA/DNA Mini Kit (Qiagen #80204) according to manufacturer’s instructions. Radiolabeled RNA was measured with a LS6500TD scintillation counter (Beckman-Coulter), and total RNA was quantified using a NanoDrop One/Onec UV-Vis Spectrophotometer (Thermo Scientific). Counts per minute (CPM) were normalized to total RNA. rRNA synthesis was measured as previously described (Ben-Sahra et al., 2013 (link)). Cells were labeled as above, and ribosomes were purified as described in Belin et al (Belin et al., 2010 ), followed by rRNA purification using the RNeasy RNA cleanup kit according to manufacturer’s instructions (Qiagen #74106), and then radiolabeled rRNA measured as above with CPM normalized to total rRNA. For quantification of rRNA and ribosomal protein per cell, RNA and protein were quantified in ribosome purifications as above, and by Bradford assay, respectively, and normalized to cells counted in parallel plates treated under the same conditions.
Valvezan A.J., Turner M., Belaid A., Lam H.C., Miller S.K., McNamara M.C., Baglini C., Housden B.E., Perrimon N., Kwiatkowski D.J., Asara J.M., Henske E.P, & Manning B.D. (2017). mTORC1 couples nucleotide synthesis to nucleotide demand resulting in a targetable metabolic vulnerability. Cancer cell, 32(5), 624-638.e5.
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