A hybridization buffer containing 45 per cent formamide, 5× SSC (750 mM NaCl and 0.075 mM sodium citrate), 20 per cent dextran sulfate, 0.1 per cent sodium dodecyl sulfate (SDS), 20 mM EDTA, 0.25 mg/ml sheared salmon sperm DNA, 0.25 mg/ml yeast RNA, and 1 per cent blocking reagent for nucleic acids (Roche, Switzerland) was used. To this buffer, the ICBM5 genome probes were added at a final concentration of 30 pg/µl for each polynucleotide probe. The samples were covered with the probe-hybridization buffer mixture and denatured for 40 min at 85°C, followed by 2 h of hybridization at 46°C. After hybridization, the samples were quickly rinsed in washing buffer I (2× SSC, 0.1 per cent SDS) at room temperature and washed in washing buffer II (0.1× SSC, 0.1 per cent SDS) for 30 min at 48°C. Finally, the samples were washed for 15 min in 1× PBS and 1 min in water and air-dried.
Yeast rna
Manufactured by Roche
Sourced in Switzerland, Spain
Yeast RNA is a type of laboratory equipment used for the extraction and purification of ribonucleic acid (RNA) from yeast cells. It is a versatile tool commonly employed in molecular biology and genetic research applications.
Automatically generated - may contain errors
Lab products found in correlation
Potato dextrose agar (pda), by Merck Group (1 mentions)
Century plus rna marker, by Thermo Fisher Scientific (1 mentions)
Sp sepharose, by GE Healthcare (1 mentions)
Hplc grade solvent, by Merck Group (1 mentions)
Ribolock rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Cl 1000 ultraviolet crosslinker, by Analytik Jena (1 mentions)
Fla 5100, by Fujifilm (1 mentions)
5 protocols using yeast rna
1
Hybridization and Washing of Genome Probes
2
Isolation and Characterization of Fungal Compounds
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The fruiting bodies of shiitake, Lentinula edodes (Berk.) Pegler, from the company Hongos Fernández Guridi (Pradejón, La Rioja, Spain) and the mealworms (Tenebrio molitor L.) were bought at local markets. The strain of Penicillium digitatum (Pers.) Sacc. was isolated in our laboratory and typified by the Spanish Type Culture Collection (CECT), Valencia, Spain. BE27 was isolated following a procedure described previously (Iglesias et al., 2015 (link)). The sources of the chemicals were described previously (Iglesias et al., 2017 ). Bovine pancreatic ribonuclease A (RNase A) and yeast RNA were purchased from Roche Diagnostics S.L. (Barcelona, Spain). SP‐Sepharose was purchased from GE Healthcare (Barcelona, Spain). Endoproteinase Glu‐C and trypsin TPCK‐treated (sequencing grade) were purchased from Merck Life Science S.r.l. (Milan, Italy). HPLC grade solvents were obtained from Merck (VWR International S.r.l., Milan, Italy). Cyanogen bromide (CNBr) was obtained from Fluka (Milan, Italy). SPDP (succinimidyl 3‐(2‐pyridyldithio) propionate) was purchased from Thermo Fisher Scientific (Rodano, Milan, Italy). Century™‐Plus RNA Markers were purchased from Fisher Scientific (Madrid, Spain). Potato dextrose agar and Potato dextrose broth media were purchased from Sigma‐Aldrich (Madrid, Spain).
3
RNA-Protein Binding Assay
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Recombinant proteins (0.25–25 μM) were incubated for 1 h on ice with 5 nM [γ-32P]-ATP-labeled RNA oligonucleotides in binding buffer (20 mM HEPES–KOH (pH 8.0), 100 mM KCl, 1.5 mM MgCl2, 5% glycerol, 0.1 μg/μl BSA, 1 μg/μl yeast RNA (Roche), 1 U/μl RiboLock RNase inhibitor (Thermo Scientific)) in 10 μl final volume. After addition of 2.5 μl of 5× loading buffer (20 mM HEPES–KOH, 100 mM KCl, 1.5 mM MgCl2, 50% glycerol, 0.1% bromophenol blue, 0.1% xylene cyanol), 4 μl of each binding reaction was loaded onto a native polyacrylamide gel (6%, 80:1 acrylamide:bis-acrylamide ratio, 0.5× TBE, 5% glycerol). Electrophoresis was carried out at 120 V for 1.5 h at 4°C. After drying, the gel was exposed on an imaging plate and scanned using the FLA-5100 phosphorimager. Bound and free RNA bands were quantified using AIDA software (Raytest). Dissociation constants were determined by nonlinear regression using GraphPad Prism (one site – specific binding) from three independent replicates.
4
Isopycnic Centrifugation of Cell Extracts
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Cs2SO4 (35% w/v final concentration) was added to supernatant or pellet fractions of the cell extracts from 10 mL cultures. After a pre-centrifugation (20 min, 16,340× g, 4 °C; Sorvall RC 5C, Hb6 rotor) for the pellet fraction, in order to remove insoluble material, both supernatants were used for the isopycnic gradient centrifugation (17 h, 50,000 rpm, 4 °C, Beckman L7-65, VTi65.1 rotor). Fractions from the bottom of the tube in 350 to 400 µL-aliquots were analyzed by UV-absorption, enzyme-linked immunosorbent assay (ELISA), Western blotting, and refractometry (Zeiss, Oberkochen, Germany). For SDS-PAGE, 100 µL aliquots were precipitated by adding 20 µL 1 mg·mL−1 yeast RNA (Roche Applied Sciences, Mannheim, Germany), 1/10 vol. 3 M Na-acetate pH 4.8 and 2 vol. ethanol, and washed twice with 70% ethanol and resuspended in 20 µL water, a quarter of which was applied per lane.
5
Northern Blot Analysis of snRNAs
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Typically, 2–3 μg of Trizol-extracted total RNA was run on a 6–8% urea-polyacrylamide gel, transferred onto Hybond-XL nylon membrane using an Owl semi-dry or tank blotter and crosslinked with UV light using the UVP CL-1000 Ultraviolet Crosslinker. Membranes were probed either with 5 × 106 cpm of [γ-32P]-ATP-labeled LNA/DNA or DNA oligonucleotides listed in Supplementary Table S1 or riboprobes produced by in vitro transcription with T7/T3/SP6 RNA polymerase in the presence of [α-32P]-UTP (major spliceosomal snRNAs in Figure 2A ). Hybridization of DNA oligonucleotide probes was carried out overnight at 37 or 42°C (probes for major spliceosomal snRNAs and U5 snRNA) in 6xSSC, 25 mM Na2HPO4/NaH2PO4 (pH 7.4), 0.5% SDS, 5× Denhardt's solution, 150 μg/ml yeast RNA (Roche). For LNA-containing oligonucleotide probes and riboprobes, hybridization was carried out overnight at 45°C and 50% formamide was included in the hybridization buffer. Membranes probed with DNA probes were washed at room temperature in 2× SSC, 0.1% SDS and 0.5× SSC, 0.1% SDS, 15 min each. For LNA and RNA probes, two additional 15 min washes with 0.1× SSC, 0.1% SDS were included, the final wash done at 60°C. Blots were exposed on imaging plates and scanned using either Typhoon FLA 9400 or Fujifilm FLA-5100.
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