RNA was first refolded by heating a solution of 5’-32P-labeled RRE-IIB (10 nM) and excess unlabeled RRE SL-IIB (200 nM) at 95 °C for 3 min and then snap cooling on ice. Refolded RNA was incubated on ice for 4 h in a solution containing the BPBA and buffer composed of 10 mM Tris, pH 7, 100 mM KCl, and 10 mM MgCl2. RNase (Ambion) was then added to the solution, which was further incubated on ice for 10 min (0.002 Units RNase V1), or 1 h (1 Unit RNase T1; 20 ng RNase A). Inactivation/precipitation buffer (Ambion) was added to halt digestion, and the RNA was pelleted by centrifugation at 13,200 rpm for 15 min. Pelleted RNA was redissolved into tracking dye and fractionated through a 12 % polyacrylamide gel containing 7.5 M urea. The gel was dried at 80 °C for 1 h and imaged by autoradiography.
Inactivation precipitation buffer
Manufactured by Thermo Fisher Scientific
The Inactivation/precipitation buffer is a solution used to deactivate and precipitate proteins or other biological molecules in a sample. It aids in the removal of interfering substances prior to further analysis or processing.
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Lab products found in correlation
5 protocols using inactivation precipitation buffer
1
RRE-IIB RNA Refolding and Footprinting
2
RNA-Protein Binding Assays with S6:S18 and S18
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For S6:S18 nuclease protection assays, the RNA–protein binding reactions identical to those used for EMSA assays above (50 μL reaction volume with S6:S18 dimer concentrations of 0, 50, 100, 300, and 600 nM) were assembled. For S18 nuclease protection assays binding reactions with S18 concentrations of 0, 25, 50, 100, 300, and 600 nM were similarly assembled. After protein-RNA incubation, each reaction was treated with 1 μL RNase A and V1 (purchased from Ambion: 20 pg and 0.002 units, respectively) for 10 min and 5 min, respectively, at 25°C. The nuclease was inactivated with inactivation/precipitation buffer (Ambion), and RNA fragments were recovered by ethanol precipitation. Precipitated RNAs were resuspended in 10 μL water and 10 μL urea loading solution (10 M urea, 1.5 mM EDTA). Ten microliters of each reaction was loaded on a 12% denaturing Acrylamide/Bis gel and run at 35W at 25°C. Partial alkaline hydrolysis reactions were generated by incubating RNA in 50 mM Na2CO3 pH 9.0, 1 mM EDTA for 10 min at 95°C. Denaturing T1 reaction was conducted by incubating 1U of RNAse T1 (Roche) in 25 mM Na Citrate pH 5.0, 5.5 M urea for 15 min at 55°C. The gel was dried and examined using a GE Healthcare STORM 820 phosphorimager and ImageQuant.
3
Hfq-mediated RNA structure probing
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32P-labeled SdsN137 or SdsN178 (∼2 nM) was incubated with purified Hfq (or equal volume of buffer) and 1 μg of yeast RNA (Ambion) in 1X RNA structure buffer (Ambion) in a total volume of 8 μl at 37°C for 15 min. Samples were mixed with RNase T1 (0.02 U, Ambion) or an equal volume of buffer and incubated at 37°C for 6 min. Inactivation/Precipitation Buffer (20 μl, Ambion) was added, and samples were placed at −80°C for ∼30 min. RNA pellets were collected by centrifugation, washed with 100 μl of 70% ethanol, air-dried and dissolved in 7 μl Gel Loading Buffer II. For the hydroxide (OH) ladder, 1 μl of 32P-labeled SdsN137 or SdsN178 in 9 μl Alkaline Hydrolysis Buffer (Ambion) was incubated 5 min at 90°C. For the RNase T1 ladder, 1 μl of 32P-labeled SdsN137 or SdsN178 in 9 μl Sequencing Buffer (Ambion) was denatured by incubating at 95°C for 1 min followed by cooling to 37°C. RNase T1 (0.1 U) was added, and the sample was incubated for 5 min at 37°C. For both ladders, the reactions were stopped by adding 12 μl of Gel Loading Buffer II. Samples (2 μl) were run on a 8% polyacrylamide-7M urea sequencing gel in 1X TBE. The gel was transferred onto Whatman filter paper, dried at 80° for 1 h, and imaged using the STORM 840.
4
RNA Structure Analysis Protocol
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We mixed 0.1 pmol (about 50,000 cpm) of labeled NsrR1 RNA in 7 µL with 2 pmol of unlabelled all1871 5′-UTR RNA, denatured for 1 min at 95 °C and chilled on ice for 5 min, followed by the addition of 1 µL of 1 mg/mL yeast RNA (Ambion AM7118) and 1 µL of 10× structure buffer (Ambion). The samples were incubated further for 15 min at 37 °C. Treatment with RNase T1, RNase A or lead(II) acetate were performed as described [10 (link)].
An alkaline ladder was obtained by incubating 0.2 pmol of 5′-labelled RNA at 95 °C for 3 min in 7.5 µL of alkaline hydrolysis buffer (Ambion) containing 1.5 µg of yeast RNA (Ambion AM7118). Reactions were stopped by the addition of 15 µL of denaturing formamide loading buffer.
RNase T1 G ladders were obtained by incubating 0.1 pmol of 5′-labelled RNA and 1 µL of 1 mg/mL yeast RNA (Ambion AM7118) in 9 µL sequencing buffer (Ambion) for 10 min at 50 °C, followed by the addition of 1 µL of 0.1 U/mL RNase T1 (Ambion AM2283) and incubation at room temperature for 15 min. Reactions were stopped by the addition of 20 µL of Inactivation/Precipitation buffer (Ambion) and incubation at −20 °C for 15 min. The precipitate was washed with 70% ethanol and resuspended in 3–7 µL of denaturing formamide loading buffer.
All samples were run on 10% polyacrylamide, 7 M urea gels and bands visualized with a Cyclone Storage Phosphor System (PerkinElmer).
An alkaline ladder was obtained by incubating 0.2 pmol of 5′-labelled RNA at 95 °C for 3 min in 7.5 µL of alkaline hydrolysis buffer (Ambion) containing 1.5 µg of yeast RNA (Ambion AM7118). Reactions were stopped by the addition of 15 µL of denaturing formamide loading buffer.
RNase T1 G ladders were obtained by incubating 0.1 pmol of 5′-labelled RNA and 1 µL of 1 mg/mL yeast RNA (Ambion AM7118) in 9 µL sequencing buffer (Ambion) for 10 min at 50 °C, followed by the addition of 1 µL of 0.1 U/mL RNase T1 (Ambion AM2283) and incubation at room temperature for 15 min. Reactions were stopped by the addition of 20 µL of Inactivation/Precipitation buffer (Ambion) and incubation at −20 °C for 15 min. The precipitate was washed with 70% ethanol and resuspended in 3–7 µL of denaturing formamide loading buffer.
All samples were run on 10% polyacrylamide, 7 M urea gels and bands visualized with a Cyclone Storage Phosphor System (PerkinElmer).
5
RNA Structural Probing by RNase Digestion
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