The sources of the chemicals were described previously [16 (link)]. Leaves from elder were harvested at Cobos de Cerrato (Palencia, Spain) in early summer. CM-Sepharose FF, Q-Sepharose FF, CM-Sepharose FF, Sepharose 6 B, and Superdex−75 HiLoad 26/60 columns were purchased from GE Healthcare (Barcelona, Spain). The acid-treated Sepharose (AT-Sepharose) was prepared as described in [66 (link)], treating the Sepharose 6 B with 0.1 N HCl at 50 °C for 3 h. The gel was then washed with water (Elix 5, Millipore) until a neutral pH was obtained, and stored in water at 4 °C until it was used. Tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin was purchased from Merk Life Science S.r.l. (Milan, Italy). Acetonitrile (CH3CN), formic acid (FA), and water (LC–MS grade) were from Fisher Scientific Italia (Milan, Italy). Century™-Plus RNA Markers were purchased from Fisher Scientific (Madrid, Spain). Rabbit reticulocyte lysate system (nuclease-treated) was purchased from Promega Biotech Iberica S.L. (Alcobendas, Madrid, Spain).
Century plus rna marker
Manufactured by Thermo Fisher Scientific
Sourced in Spain
The Century™-Plus RNA Markers are a set of pre-stained RNA size standards that allow for the convenient size determination of RNA molecules during gel electrophoresis. The markers cover a range of sizes from 0.2 to 9.5 kilobases.
Automatically generated - may contain errors
Lab products found in correlation
Thermopol buffer, by New England Biolabs (2 mentions)
Typhoon trio laser scanner, by GE Healthcare (2 mentions)
Rnase a, by Thermo Fisher Scientific (2 mentions)
Storm 860 molecular imager, by GE Healthcare (2 mentions)
Sepharose 6b, by GE Healthcare (1 mentions)
Cm sepharose ff, by GE Healthcare (1 mentions)
Q sepharose ff, by GE Healthcare (1 mentions)
Potato dextrose agar (pda), by Merck Group (1 mentions)
Yeast rna, by Roche (1 mentions)
Sp sepharose, by GE Healthcare (1 mentions)
Hplc grade solvent, by Merck Group (1 mentions)
6 protocols using century plus rna marker
1
Purification and Characterization of Elderberry Compounds
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
Assessing RNase Contamination in Enzymes
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Contaminating RNase Activity in purified GT-MMLV and GT-Taq was assayed by monitoring degradation of an RNA gel ladder. The reaction solution included the test enzyme at its storage concentration (1 μL), 0.5 μL of Century-Plus RNA Marker (Thermo Fisher Scientific #AM7145), 2 μL of 10X ThermoPol Buffer (NEB #B9004), and nuclease-free water to a final volume of 20 μL. As a positive control, 1 μL of 1 pg/mL RNase A (Thermo Fisher Scientific EN0531) stock solution was added to OneTaq®. The solutions were incubated at 37°C for 30 min, at which point the reaction was quenched by the addition of an equal volume of 2X loading buffer and dye (95% formamide, 0.025% bromophenol blue, 0.025% xylene cyanol, 5 mM EDTA pH 8.0). The quenched samples were evaluated for ladder degradation by gel electrophoresis using 16 cm × 16 cm 10% polyacrylamide gels with 8 M urea. Gels were run in 1X TBE (Tris/boric acid/EDTA pH 8.0) at 14 W and 300–400 V for at least 30 min prior to loading samples and running for an additional 1.5 h. Gels were stained with ethidium bromide and scanned on the Typhoon Trio+ laser scanner (GE Healthcare) using a channel with 532 nm excitation and a 610 nm emission filter. Smearing of the ladder indicated RNase contamination. The presence of intact bands confirmed the absence of RNase in in-house produced enzymes.
4
Assessing RNase Contamination in Enzymes
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Contaminating RNase Activity in purified GT-MMLV and GT-Taq was assayed by monitoring degradation of an RNA gel ladder. The reaction solution included the test enzyme at its storage concentration (1 μl), 0.5 μl of Century-Plus RNA Marker (Thermo Fisher Scientific, #AM7145), 2 μl of 10× ThermoPol Buffer (New England Biolabs, #B9004), and nuclease-free water to a final volume of 20 μl. As a positive control, 1 μl of 1 pg/ml RNase A (Thermo Fisher Scientific, #EN0531) stock solution was added to OneTaq®. The solutions were incubated at 37 °C for 30 min, at which point the reaction was quenched by the addition of an equal volume of 2× loading buffer and dye (95% formamide, 0.025% bromphenol blue, 0.025% xylene cyanol, 5 mm EDTA, pH 8.0). The quenched samples were evaluated for ladder degradation by gel electrophoresis using 16 × 16-cm 10% polyacrylamide gels with 8 m urea. Gels were run in 1× TBE (Tris/boric acid/EDTA, pH 8.0) at 14 W and 300–400 V for at least 30 min prior to loading samples and running for an additional 1.5 h. Gels were stained with ethidium bromide and scanned on the Typhoon Trio+ laser scanner (GE Healthcare) using a channel with 532-nm excitation and a 610-nm emission filter. Smearing of the ladder indicated RNase contamination. The presence of intact bands confirmed the absence of RNase in in-house produced enzymes.
5
Catalytic Cleavage of aac(6')-Ib mRNA
6
Evaluating aac(6')-Ib mRNA Cleavage by EGSs
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The EGSs were tested by preincubating 5′-end-labeled aac(6’)-Ib mRNA (5 pmol) and the EGS (10 pmol) at 25°C for 30 min in a volume of 3 µl before adding this mixture to a solution containing 2.5 pmol of M1 RNA, 70 pmol of C5 protein, 20 mM HEPES-KOH (pH 8.0), 400 mM ammonium acetate, 10 mM magnesium acetate, and 5% glycerol that had been preincubated at 37°C for 15 min in a final volume of 7 µl [54 (link)]. After combining both solutions the mix was incubated at 37°C for the times indicated. The reaction was stopped by the addition of 1 volume of phenol-chloroform followed by ethanol precipitation. The pellet was resuspended in 10 µl DEPC-treated water, mixed with 10 µl of 2 x gel loading buffer, and analyzed by 5% denaturing TBE-PAGE along with labeled RNA Century Marker or RNA Century Marker-Plus (ThermoFisher) as described before [31 (link)]. Fluorescence was detected on a Storm 860 Molecular Imager (Molecular Dynamics) and the signal in each band was quantified using Image J [55 ].
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