Structure probed RNA was fragmented at 95 °C for 3.5 min in alkaline hydrolysis buffer (Ambion). As fragmentation results in 5′OH, and is hence ligation incompatible, it does not interfere with the downstream library preparation process. Fragmented RNA was then purified using RiboMinus concentration module (Life Technologies), using the modified protocol for RNAs that are <200 bases. The RNA was eluted in 12 µl of nuclease free water and concentrated to 2 µl using a vacuum centrifuge. The RNA was then ligated to 5′ adapter from NEBNext Multiplex Small RNA Library Prep Set for Illumina using T4 RNA ligase1 (T4 RNA ligase buffer, 1 mM ATP, 10% PEG, 10% DMSO) at 16 °C overnight. The 5′ adapter ligated RNAs were then purified through a 6% TBE urea PAGE gel and size selected for 50–200 bases. The RNA was then ligated to 3′ adapter, reverse transcribed, and PCR amplified using the NEBNext Multiplex Small RNA Library Prep Set (New England Biolabs) for Illumina using manufacturer’s instructions. Eighteen cycles of PCR amplification were typically performed for each library.
Alkaline hydrolysis buffer
Manufactured by Thermo Fisher Scientific
Alkaline hydrolysis buffer is a laboratory solution used to facilitate the breakdown of organic compounds through the process of alkaline hydrolysis. This buffer maintains a high pH environment to promote the hydrolysis reaction.
Automatically generated - may contain errors
Lab products found in correlation
Rnase t1, by Thermo Fisher Scientific (7 mentions)
T4 polynucleotide kinase, by New England Biolabs (2 mentions)
Inactivation precipitation buffer, by Thermo Fisher Scientific (2 mentions)
Rnase v1, by Thermo Fisher Scientific (2 mentions)
Nebnext multiplex small rna library prep set, by Illumina (1 mentions)
Ribominus concentration module, by Thermo Fisher Scientific (1 mentions)
Nebnext multiplex small rna library prep set, by New England Biolabs (1 mentions)
Yeast trna, by Thermo Fisher Scientific (1 mentions)
Rna structure buffer, by Thermo Fisher Scientific (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
Typhoon phosphoimager, by GE Healthcare (1 mentions)
Fla 3000 series, by Fujifilm (1 mentions)
Imagequant software, by GE Healthcare (1 mentions)
Storm 860 molecular imager, by GE Healthcare (1 mentions)
Yeast rna, by Thermo Fisher Scientific (1 mentions)
Cyclone storage phosphor system, by PerkinElmer (1 mentions)
Rna sequencing buffer, by Thermo Fisher Scientific (1 mentions)
Rnase t1 kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using alkaline hydrolysis buffer
1
RNA Fragmentation and Library Prep
2
Enzymatic Probing of RNA Structure
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
5′-end labeled transcripts were prepared as described for the EMSAs. The enzymatic probing was carried out as previously described (Sievers et al., 2014 (link)), with some deviations. Briefly, for the alkaline hydrolysis ladder, 0.2 pmol of labeled RNA was mixed with alkaline hydrolysis buffer (Ambion) and 10 μg of yeast tRNA (Ambion) in a total volume of 10 μL and incubated at 95°C for 5 min; for T1 control sample, 0.2 pmol of labeled RNA was denatured and incubated with 0.01 U of T1 RNase (Ambion) for 5 min. Structure probing RNA interactions were incubated at 37°C for 1 h before treating the samples with the indicated cleaving agent: 0.01 U T1 RNase for 5 min and 0.0015 U V1 RNase (Ambion) for 2 min. Control samples were prepared likewise (except for the cleaving agents) and incubated at 37°C for the duration of the experiment. Samples were placed on ice and mixed with 2× loading buffer type II (Ambion). Five μL of each sample was separated on an 8% denaturing polyacrylamide gel. RNA bands were visualized and analyzed as described for the northern blotting experiments.
3
RNA-inhibitor Interaction Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
r(AUUCU)11 was radioactively labelled at the 5′ end with [γ-32P] ATP (Perkin Elmer) and T4 polynucleotide kinase (New England Biolabs) using standard methods and gel purified to homogeneity51 . The RNA was folded by incubation at 60 °C for 5 min in 1 × RNA Structure Buffer (Ambion) followed by slow cooling to room temperature. Serially diluted concentrations of the inhibitor was added to the RNA solution and incubated at room temperature for 15 min. RNase V1 (Ambion) was added to the RNA-inhibitor complex to a final concentration of 5 μU μl−1 and the samples were incubated at room temperature for 60 min. RNase V1 was then inactivated by heating at 95 °C for 1 min, and cleavage products were separated on a denaturing 20% polyacrylamide gel. A hydrolysis ladder was prepared by using Alkaline Hydrolysis Buffer (Ambion) and the manufacturer's protocol.
4
RNA structure probing by RNase T1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
5΄-end-labeled RNA in 0.5× TE buffer was heated at 95°C for 3 min and snap-cooled on ice for 10 min. A total of 1 μl of RNA solution was added to 9 μl buffer containing 250 mM KCI or LiCI and Tris–HCI (pH 7.4) for 15 min at room temperature. A total of 0.1 units RNase T1 (Ambion) was added to the RNA and incubated at room temperature for 5 min. Reactions were quenched with 10 μl phenol and then phenol–chloroform extractions were performed. To generate a uniform ladder, 1 μl RNA was incubated with 9 μl alkaline hydrolysis buffer (Ambion) for 3 min at 95°C. Products were separated using appropriate percentage of denaturing 29:1 acrylamide;bisacrylamide gel by electrophoresis. Gels were dried and visualized using a Typhoon PhosphoImager (Molecular Dynamics).
5
In vitro Structure Probing of flaA Leaders
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vitro structure probing of flaA WT and flaA M1/M2 leaders with RNase T1 and lead(II) acetate was performed as previously described68 (link). For each reaction, 0.1 pmol of a labelled flaA leader variant was denatured for 1 min at 95 °C and chilled on ice for 5 min. One microgram yeast tRNA as competitor and 10 × RNA Structure Buffer was added (provided together with RNase T1, Ambion). Unlabelled recombinant C. jejuni CsrA protein was then added at 0-, 20-, 50- or 100-fold molar excess. After incubation for 15 min at 37 °C, 2 μl RNase T1 (0.01 U μl−1) or 2 μl freshly prepared lead(II)-acetate solution (25 mM) were added and reactions were incubated for 3 min or 90 s, respectively. As a control, ∼0.1 pmol labelled RNA with 100-fold excess CsrA was also prepared without nuclease/lead(II) treatment. The reactions were stopped by addition of 12 μl Gel loading buffer II (#AM8546G, Ambion). For RNase T1 ladders, ∼0.1 pmol labelled RNA was denatured in 1 × Structure Buffer for 1 min at 95 °C and afterwards incubated with 0.1 U μl−1 RNase T1 for 5 min. The OH ladder was generated by incubation of ∼0.1 pmol labelled flaA WT leader RNA in 1 × alkaline hydrolysis buffer (Ambion) for 5 min at 95 °C. Ladders and samples were then separated on 10% (v/v) PAA/7M urea gels in 1 × TBE buffer. Gels were dried, exposed to a screen and analysed using a PhosphorImager (FLA-3000 Series, Fuji).
6
Structural Probing of lrp1 and lrp2 mRNAs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Structure probing was performed using 0.1 pmol of mRNA (lrp1 and lrp2) in 10 μl reactions as previously described by Sharma et al. (40 (link)). 5′-end-labeled RNA was denatured for 1 min at 95°C followed by incubation on ice for 5 min and hybridized with five-fold excess of cold sRNA for 60 min at 37°C in the presence of 1 μg of yeast tRNA, 100-nM Hfq protein and 1x structure buffer. The RNA mixture was digested with a final concentration of 5-mM lead(II) (Fluka) or 0.002 units of RNase T1 (Ambion) for 1.5 or 3 min at 37°C. An RNase T1 ladder was generated by incubating 0.2 pmol of denatured mRNA with 0.1 units of RNase T1 in 1x sequencing buffer (Ambion) for 5 min at 37°C. The OH ladder was obtained by incubating 0.2 pmol of mRNA in alkaline hydrolysis buffer (Ambion) for 5 min at 95°C. Reactions were stopped by adding 12 μl loading buffer II (95% v/v formamide, 18-mM EDTA, 0.025% SDS, xylene cyanole, bromophenol blue; Ambion). Samples were denatured for 3 min at 95°C and run on 6% polyacrylamide/7M urea sequencing gels in 1x TBE buffer at 65 W for 120 min for lrp1 and 60 min for lrp2. Gels were dried and analyzed using Storm 860 Molecular Imager and ImageQuant software (GE Healthcare).
7
Hfq-mediated RNA structure probing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
8
RNA Structure Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
9
Cleavage Product Length Determination
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!