miRNAs, DNA probes labeled with AlexaFluor 594, and IDTE buffer (10 mM Tris, 0.1 mM ethylenediaminetetraacetic acid, pH 7.5) were purchased from Integrated DNA Technologies (Coralville, IA). miRNAs and probes (Table S1 ) were reconstituted in 1x IDTE and stored at −20 °C. Pluronic F-127 (PF-127), magnesium chloride, ammonium acetate, and rhodamine 6G were obtained from Millipore Sigma (Burlington, MA). Tris-HCl and glycine were purchased from Fisher Scientific (Pittsburgh, PA). All solutions were prepared with 18.2 MΩ·cm ultrapure water from an ELGA LabWater Purelab Classic (High Wycombe, UK).
Idte buffer
Manufactured by Integrated DNA Technologies
Sourced in United States
IDTE buffer is a buffer solution designed for use in molecular biology applications. It is a commonly used buffer for the dilution and storage of DNA and RNA samples. The core function of IDTE buffer is to provide a stable and controlled environment for the preservation and handling of nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
Glycine, by Thermo Fisher Scientific (2 mentions)
Ammonium acetate, by Merck Group (2 mentions)
Tris hcl, by Thermo Fisher Scientific (2 mentions)
Rhodamine 6g, by Merck Group (2 mentions)
Dna probes labeled with alexafluor 594, by Integrated DNA Technologies (2 mentions)
Primerquest tool, by Integrated DNA Technologies (2 mentions)
Dna oligonucleotides, by Thermo Fisher Scientific (2 mentions)
Rna oligonucleotides, by Integrated DNA Technologies (2 mentions)
Oligoanalyzer tool, by Integrated DNA Technologies (2 mentions)
Nebuilder tool, by New England Biolabs (1 mentions)
Qubit dsdna high sensitivity assay kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using idte buffer
1
Fluorescent Labeling of miRNAs
2
Oligonucleotide Design for miRNA Analyses
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RNA oligonucleotides of unedited miR-379, edited miR-379, miR-380, unedited miR-411, edited miR-411, and miR-758 were purchased from Integrated DNA Technologies, dissolved in IDTE buffer, pH 7.5 (Integrated DNA Technologies), and then diluted in 10-fold dilution series for RT-qPCR. For experiments with yeast RNA background, 100 ng total yeast RNA (#AM7118, Invitrogen, Thermo Scientific) was added during RT sample preparation. DNA oligonucleotides were purchased from Invitrogen. Two-tailed RT primers were designed based on a hairpin sequence published by Androvic et al. (2017) (link) with hemiprobes designed to bind unedited or edited miR-379, and primer arms optimized to prevent the formation of unwanted secondary structures. Secondary structures of RT primers as well as secondary structures and dimers for qPCR primers were calculated using the OligoAnalyzer tool (Integrated DNA Technologies). ZEN/Iowa Black FQ double-quenched FAM-coupled miR-379 hydrolysis probe was designed using the PrimerQuest tool (Integrated DNA Technologies) and purchased from Integrated DNA Technologies. Primers for pri-miR-379 RT-PCR were based on those published by Kawahara et al. (2008) (link), and primers for cloning were designed using the NEBuilder tool (New England Biolabs). All RNA and DNA oligonucleotide sequences are listed in Supplemental Tables 2 and 3 .
3
Amplicon Pooling for Sequencing
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The expected size of amplicons was confirmed by electrophoresis on 0.6% agarose gels (run at 60 V) and 1 μl of the reaction mixture was used to measure the amplicon concentration with the Qubit dsDNA High Sensitivity Assay kit (ThermoFisher, Waltham, USA).
All amplicons that would be sequenced with the same index sequence (either all PCRs of a specific proband or non-overlapping amplicons of different probands) were diluted to 10 ng/μl in IDTE buffer (Integrated DNA Technologies, Coralville, USA). The volume of each diluted PCR added to the pool was proportional to its size, for a final pool of at least 130 μl in total volume.
All amplicons that would be sequenced with the same index sequence (either all PCRs of a specific proband or non-overlapping amplicons of different probands) were diluted to 10 ng/μl in IDTE buffer (Integrated DNA Technologies, Coralville, USA). The volume of each diluted PCR added to the pool was proportional to its size, for a final pool of at least 130 μl in total volume.
4
miRNA Labeling and Quantification
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Reagents miRNAs, DNA probes labeled with AlexaFluor 594, and IDTE buffer (10 mM Tris, 0.1 mM ethylenediaminetetraacetic acid, pH 7.5) were purchased from Integrated DNA Technologies (Coralville, IA). miRNAs and probes (Table S1) were reconstituted in 1x IDTE and stored at -20 °C. Pluronic F-127 (PF-127), magnesium chloride, ammonium acetate, and rhodamine 6G were obtained from Millipore Sigma (Burlington, MA). Tris-HCl and glycine were purchased from Fisher Scientific (Pittsburgh, PA). All solutions were prepared with 18.2 MΩ•cm ultrapure water from an ELGA LabWater Purelab Classic (High Wycombe, UK).
5
Quantitative Analysis of Edited miRNA
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RNA oligonucleotides of unedited miR-379, edited miR-379, miR-380, unedited miR-411, edited miR-411, and miR-758 were purchased from Integrated DNA Technologies, dissolved in IDTE buffer, pH 7.5 (Integrated DNA Technologies, Coralville, IA), and then diluted in 10-fold dilution series for RT-qPCR. For experiments with yeast RNA background, 100 ng total yeast RNA (#AM7118, Invitrogen, Thermo Scientific, Vilnius, Lithuania) was added during reverse transcription (RT) sample preparation. DNA oligonucleotides were purchased from Invitrogen. Two-tailed RT primers were designed based on a hairpin sequence published by Androvic et al. (Androvic et al. 2017) with hemiprobes designed to bind unedited or edited miR-379, and primer arms optimized to prevent the formation of unwanted secondary structures. Secondary structures of RT primers as well as secondary structures and dimers for qPCR primers were calculated using the OligoAnalyzer tool (Integrated DNA Technologies). ZEN/Iowa Black FQ double-quenched FAM-coupled miR-379 hydrolysis probe was designed using the PrimerQuest tool (Integrated DNA Technologies) and purchased from Integrated DNA Technologies. Primers for pri-miR-379 RT-PCR were based on those published by Kawahara et al. (Kawahara et al. 2008) . All RNA and DNA oligonucleotide sequences are listed in Supplementary Table 1 and2.
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