(U)30 oligonucleotide (IDT) was radiolabelled with a 5′ phosphate using [γ-32P] ATP and PNK enzyme (ThermoFisher). 3′ end-labeled oligonucleotide (U)30 was prepared using 5-[32P]-pCp and T4 RNA ligase (NEB). Labelled oligonucleotides were each separated from free nucleotides on mini oligo Quick spin columns (Roche) and subsequently gel purified. For nuclease assays, 1 μM of WT or mutant recombinant NYN (238–510) was incubated with 10 pmol of cold (U)30 RNA, 0.1 pmol of radiolabelled (5′ or 3′) (U)30 in 10 μl reaction volumes containing 20 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol and 1 mM DTT. Reactions were also supplemented with 3 mM MnCl2 or 3 mM MgCl2 as described in the figure legends. Reactions were carried out at room temperature for up to 60 min. RNA at each time point was ethanol precipitated and resuspended in 10 μl loading buffer II (ThermoFisher). Samples were boiled at 95°C for 10 min and resolved by electrophoresis on a 15% polyacrylamide and 8 M urea gel and visualized by phosphorimaging (Storm, GE Life Sciences). RNA ladders were prepared by partial alkaline hydrolysis of 5′ end-radiolabeled oligonucleotide (U)30 RNA. This was performed in a 5 μl reaction mixture containing 0.1 pmol RNA and 66.7 mM (NaHCO3/Na2CO3, pH 9.5) incubated at 95°C for 5 min. Reactions were terminated by adding 10 μl of loading buffer II (ThermoFisher).
Loading buffer 2
Manufactured by Thermo Fisher Scientific
Sourced in United States
Loading buffer II is a solution used in molecular biology applications to prepare samples for gel electrophoresis. It is designed to ensure the proper loading and migration of DNA, RNA, or protein samples into agarose or polyacrylamide gels.
Automatically generated - may contain errors
Lab products found in correlation
Omnidoc imager, by Cleaver Scientific (2 mentions)
Megascript t7 transcription kit, by Thermo Fisher Scientific (2 mentions)
Monarch pcr and dna cleanup kit, by New England Biolabs (2 mentions)
Polyacrylamide tbe urea gel, by Thermo Fisher Scientific (2 mentions)
Rnasin plus rnase inhibitor, by Promega (1 mentions)
Sequenase 2.0 dna sequencing kit, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Storm 840 phosphorimager, by GE Healthcare (1 mentions)
Neb3 buffer, by New England Biolabs (1 mentions)
Rnasin rnase inhibitor, by Promega (1 mentions)
Typhoon fla 9500, by GE Healthcare (1 mentions)
Ultrahyb oligo, by Thermo Fisher Scientific (1 mentions)
Northern max gly kit, by Thermo Fisher Scientific (1 mentions)
Odyssey northern blot analysis protocol, by LI COR (1 mentions)
Ultrahyb oligo buffer, by Thermo Fisher Scientific (1 mentions)
Odyssey imager, by LI COR (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
10 protocols using loading buffer 2
1
Radiolabeling and Nuclease Assay for (U)30 RNA
2
In vitro Transcription and Xrn1 Treatment of 3'UTRs
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A DNA fragment corresponding to the RaTV 3′UTR under the T7 promoter was synthesized and cloned into the pIDT vector (IDT). Plasmids containing PCV 3′UTR and GFP gene fragments have been described previously [38 (link)]. For in vitro transcription, plasmids were first linearized by restriction digest and purified using a Monarch PCR and DNA Clean-up Kit (NEB). 3′UTRs were in vitro transcribed from 500 ng of plasmid DNA using a MEGAscript T7 Transcription Kit (Invitrogen). RNA was purified by LiCl precipitation and examined by electrophoresis in a 1 % denaturing agarose gel. RNA was then refolded in NEB3 buffer (85 °C for 5 min) and gradually cooled to 28 °C. For Xrn1 treatment, refolded RNA (1 µg) was incubated with 1 U Xrn1 (NEB) and 10 U RppH (NEB) in 20 µl of reaction mixture containing 1× NEB3 buffer (NEB) and 1 U µl–1 RNasin Plus RNase Inhibitor (Promega) for 2 h at 28 °C. The reaction was stopped by adding 20 µl of Loading Buffer II (Ambion), heating for 5 min at 85 °C and placing on ice. The denatured RNA samples were loaded into 6 % polyacrylamide TBE-Urea gels (Invitrogen), and electrophoresis was performed for 90 min in 1× TBE. The gels were stained with ethidium bromide and imaged using an OmniDoc imager (Cleaver Scientific).
3
Hfq and SgrS Regulation of manX Expression
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Toeprinting assays were performed using unlabeled manXATG and P32-end-labeled primer in the presence and absence of Hfq and SgrS following the previously published protocol (39 ). For each reaction, 2 pmol of manX RNA and 1.6 pmol of end-labeled primer (O-JH119) were heated for one min at 95°C in toeprint buffer (20 mM Tris–HCl pH 7.5, 50 mM KCl, 0.1 mM EDTA, and 1mM DTT). The mixture was chilled in ice for 5 min, followed by addition of MgCl2 and dNTPs (10 and 1 mM respectively, final concentrations). Purified Hfq and in vitro synthesized SgrS RNA were also added to the appropriate reactions and incubated at 37°C for 10 min. Next, ribosomes (1.3 pmol, NEB) were added to this reaction mixture, and the incubation was continued at 37°C for 5 min. Thirteen picomoles of fMet-tRNA (Sigma) was added to this reaction and cDNAs were synthesized using SuperScript III reverse transcriptase (Invitrogen). The reaction was stopped by adding 10 μl of loading buffer II (Ambion). The reaction products were analyzed on an 8% polyacrylamide-urea gel. Sequencing ladders were generated using Sequenase 2.0 DNA sequencing kit (Affymetrix).
4
Characterizing sRNA-Ribosome Interactions
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Gel retardation assays were performed (26 (link)), and 0.4 pmol of labelled sbi mRNA or RNAIII were incubated with various concentrations (from 1.6 to 50 pmols) of unlabelled RNAs. The toeprint assays were performed as previously described (26 (link)). The annealing mixtures used contained 0.2 pmol of sbi mRNA and 1 pmol of labelled SBIrevTR primer in a reaction buffer of 10 mM Tris-acetate, pH 7.5, 60 mM NH4Cl and 1 mM dithiothreitol (DTT). For the competition assays, various concentrations of SprD, RNAIII or RNAIII mutants were added before the purified Escherichia coli 70S ribosomes. Ribosomes were activated for 15 min at 37°C and diluted in the same reaction buffer in the presence of 1 mM MgCl2. Next, 4 pmols of ribosomes were added and incubated for 5 min, then the MgCl2 was adjusted to 10 mM and the reactions were incubated for 5 min. In all, 10 pmols of uncharged transfer RNAfMet (tRNAfMet) were then added for 15 min. Complementary DNAs were synthesized with 2 UI of AMV RT (Biolabs) for 15 min at 37°C. Reactions were ended by the addition of 10 µl of loading buffer II (Ambion). The complementary DNAs were separated in 8% denaturating polyacrylamide gel electrophoresis. Gels were dried and visualized using a STORM 840 Phosphorimager (Molecular Dynamics).
5
Toeprint Assay for mRNA-Ribosome Interactions
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The toeprint assays were performed as previously described (20 (link)), but with modifications. Annealing mixtures containing 13 nM of yabJ-spoVG mRNA with either 67 nM of labeled Toeprint_spoVG or Toeprint_yabJ primers were incubated with a buffer (20 mM Tris–HCl, pH 7.5, 60 mM NH4Cl) for 2 min at 90°C followed by 1 min at RT. Renaturation was realized in the presence of 10 mM MgCl2 at 25°C for 20 min. For the assays in the presence of SprX, various concentrations of SprX were added before the purified E. coli 70S ribosomes. The ribosomes were reactivated for 15 min at 37°C and diluted in the reaction buffer in the presence of 1 mM MgCl2. 33 nM 70S were added in each assay, incubated for 5 min and the MgCl2 was adjusted to 10 mM. After 5 min, 0.83 µM uncharged tRNAfMet was added and this was incubated for 15 min. cDNAs were synthesized with 4 U of AMV RT (Biolabs) for 15 min. Reactions were ended by the addition of 15 µl of loading buffer II (Ambion). The cDNAs were loaded and separated onto 8% polyacrylamide/8M urea gels. Sequencing ladders were generated with the same 5′-end-labeled primer.
6
In Vitro 3'UTR Transcription and RNA Analysis
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Plasmids were linearised by restriction digest and purified using Monarch PCR and DNA Clean-up Kit (NEB, USA). 3’UTRs were in vitro transcribed from 1 μg of linearised plasmids using MEGAscript T7 Transcription Kit (Invitrogen, USA) according to the manufacturer’s recommendations. RNA was purified by LiCl precipitation and analysed by electrophoresis in a 1.2% denaturing agarose gel. RNA was then refolded in NEB3 buffer by heating at 85 °C for 5 min followed by gradual cooling to 28 °C. The refolded RNA (1 μg) was incubated with 1U XRN1 (NEB, USA) and 10U RppH (NEB, USA) in 20 μL of reaction mixture containing 1x NEB3 buffer (NEB, USA) and 1 u/μL RNasin RNase Inhibitor (Promega, USA). Incubation was performed for 2 h at 28 °C. The reaction was stopped by adding 20 μL of Loading Buffer II (Ambion, USA), heating for 5 min at 85 °C and placing on ice. The entire volume was then loaded into 6% polyacrylamide TBE-Urea gels (Invitrogen, USA), and electrophoresis was performed for 90 min in 1xTBE. Gels were stained with ethidium bromide and documented using an Omnidoc imager (Cleaver Scientific, UK).
7
Toeprint Assay for Ribosome Binding
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The toeprint assays were performed as previously (12 (link)), with modifications. Annealing mixtures containing 0.2 pmol of ecb mRNA and 1 pmol of labeled primer 34 in a reaction buffer (100 mM Tris–HCl pH 7.5, 300 mM NH4Cl, 5 mM DTT) were incubated 2 min at 90°C, followed by 1 min incubation on ice. RNA refolding was performed in the presence of 10 mM MgCl2 for 10 min at room temperature. SprX2 and SprX2mutL1 RNAs were added at the specified concentrations before the addition of purified E. coli 70S ribosomes. The ribosomes were reactivated for 15 min at 37°C and diluted in presence of 1mM MgCl2. 1 pmol of 70S ribosomes were added in each assay and incubated 5 min at 37°C. The concentration of MgCl2 was adjusted to 10 mM. After 10 min at 37°C, 10 pmol of uncharged transfer RNAfMet (MP Biomedicals) was added and incubated 5 min at 37°C. Complementary DNAs were synthesized with 2 UI of AMV RT (Biolabs) for 15 min at 37°C. Reactions were ended by the addition of 15 μl of loading buffer II (Ambion). The cDNAs were separated in 8% denaturing polyacrylamide gel electrophoresis. Gels were dried and visualized using Typhoon FLA 9500 (GE Healthcare).
8
Structural Analysis of SprX-yabJ-spoVG167 mRNA
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Structural assays were performed as previously described (34 (link)). SprX was prepared by incubating 14 pmol of unlabeled RNA in a buffer (10 mM Tris–HCl, pH 7.5, 60 mM NaCl, 1 mM EDTA) for 10 min at 25°C. The yabJ-spoVG167 mRNA was prepared by incubating 1 pmol of labeled RNA in the aforementioned buffer. MgCl2 was added to obtain a final concentration of 2.5 mM and this was then incubated for 10 min at 25°C. Preparation for structural analysis of the duplexes between SprX and yabJ-spoVG167 mRNA was done by incubating 0.2 pmol of labeled RNA with 10 pmol of unlabeled RNA for 15 min at 25°C. Cleavages with S1 nuclease (0.063 U/µl),V1 RNase (6.25 × 10−7 U/µl) and 1.25 mM lead acetate were carried out for 10 min at 25°C in the presence of 1 μg of total yeast tRNA. For sequencing, T1 at 0.038 U/µl and U2 at 0.0015 U/µl were used. The reactions were precipitated, and the pellets dissolved in loading buffer II (Ambion). The samples were denatured for 10 min at 65°C before separation on 8% polyacrylamide/8M urea gels. Gels were dried and visualized (Typhoon FLA 9500).
9
Comprehensive mRNA and sRNA Analysis
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For mRNA analysis, total RNA (15 μg) in glyoxal loading dye was run on a 1% TBE-agarose gel and then transferred to a nylon membrane using the NorthernMax-Gly Kit (Life Technologies, AM1946). For sRNA analysis, total RNA (2 μg) in Loading Buffer II (Life Technologies, AM8546G) was run on a 10% TBE-urea polyacrylamide gel and then transferred to a nylon membrane (GE Healthcare Life Sciences, RPN119B). For all blotting, RNA was subjected to UV cross-linking after transfer, the blot was pre-hybridized in ULTRAhyb-Oligo (Life Technologies, AM8663) at 65 °C for at least 30 min, and then hybridized overnight at 65°C. In some cases, hybridizations were performed using DNA probes (Table S2 ) 5′ end labeled with an IR dye. Otherwise, northern blotting was performed with RNA probes transcribed from PCR-derived templates (Table S2 ) with T7 promoters by using biotin-16-UTP and T7 RNA polymerase (Promega, P2075) according to the manufacturer's instructions (see above). All blots were washed and imaged as described in the Odyssey northern blot analysis protocol (Licor).
10
Quantitative Northern Blotting Protocol
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To prepare samples for Northern blotting, total RNA was mixed 1:2 in Loading Buffer II (Life Technologies). RNA was separated on a 10% Tris-borate-EDTA (TBE)–urea gel, run at 200 V for 50 to 60 min in 1× TBE. Transfer to a positively charged nylon membrane was performed using the TransBlot Turbo transfer system (7 min at 1.3 A; Bio-Rad).
Following a wash in 6× saline sodium citrate (SSC) for 2 min, the nylon membrane was subjected to UV cross-linking, followed by another wash in 1× SSC for 1 min. The membrane was then prehybridized for at least 30 min in ULTRAhyb-Oligo buffer (Life Technologies) at 65°C. Overnight hybridization was performed at 65°C with the appropriate riboprobe and a 5S DNA probe (IR800-5S). The membrane was subsequently washed two times for 5 min each time and two times for 15 min each time in low- and high-stringency wash buffer, respectively, according to the Odyssey Northern blot analysis protocol instructions (LI-COR). Fluorescence imaging was conducted using the Odyssey imager (LI-COR). Band quantifications were performed using ImageStudio (version 5.0) software (LI-COR). Statistical analysis was performed using GraphPad Prism (version 7) software.
Following a wash in 6× saline sodium citrate (SSC) for 2 min, the nylon membrane was subjected to UV cross-linking, followed by another wash in 1× SSC for 1 min. The membrane was then prehybridized for at least 30 min in ULTRAhyb-Oligo buffer (Life Technologies) at 65°C. Overnight hybridization was performed at 65°C with the appropriate riboprobe and a 5S DNA probe (IR800-5S). The membrane was subsequently washed two times for 5 min each time and two times for 15 min each time in low- and high-stringency wash buffer, respectively, according to the Odyssey Northern blot analysis protocol instructions (LI-COR). Fluorescence imaging was conducted using the Odyssey imager (LI-COR). Band quantifications were performed using ImageStudio (version 5.0) software (LI-COR). Statistical analysis was performed using GraphPad Prism (version 7) software.
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