A DNA template with SP6-promoter for transcribing a radiolabeled RNA probe that can hybridize to a positive sense strand of RCNMV RNA1 3′ UTR (nts 3605 to 3800) was prepared by PCR with the following composition and conditions: An amount of 50 µL reaction with GoTaq G2 green master mix (1×), R1.3UTR.for (0.2 µM, 5′-TCG GAC CCT GGG AAA CAG GT-3′), R1.3UTR.SP6.rev (0.2 µM, 5′-GATATTTAGGTGACACTATAG AGGTATGCGCCCTCTGAGC-3′), pRC169 as template (10 ng); initial denaturation at 95°C for 2 min; 25 cycles of denaturation at 95°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 30 sec; final extension at 72°C for 5 min. The underlined bases in the primer sequence represent SP6 promoter sequence. The amplified product was purified using QIAquick PCR Purification kit (Qiagen #28104) and used as a template for making a radiolabeled probe using MEGAscript SP6 Transcription kit (Invitrogen #AM1330) with the following reaction composition: An amount of 2 µL 10× reaction buffer, 2 µL 5mM AUG mix, 2.5 µL 0.1 mM CTP, 50 ng DNA template, 0.5 µL RNase OUT (Invitrogen #10777019), 2 µL SP6 enzyme, 2.5 µL CTP (ɑ-32P; PerkinElmer #BLU008X250UC). The reaction was incubated at 37°C for 3 h followed by DNase treatment with 1 µL Turbo DNase at 37°C for 15 min and a radiolabeled RNA probe was purified using Micro Bio-spin 30 columns (Bio-Rad #732-6251) and stored at −20°C.
Micro bio spin 30 column
Manufactured by Bio-Rad
Sourced in United States
The Micro Bio-Spin 30 Columns are size exclusion chromatography columns designed for rapid, efficient purification of small biomolecules, such as nucleic acids and proteins, from complex samples. The columns utilize a proprietary resin to separate molecules based on their size, allowing for the removal of unwanted contaminants and buffer exchange.
Automatically generated - may contain errors
Lab products found in correlation
Riboprobe system, by Promega (5 mentions)
Qiaquick pcr purification kit, by Qiagen (3 mentions)
35s utp, by Cytiva (3 mentions)
35s utp, by PerkinElmer (2 mentions)
T7 polymerase, by Promega (2 mentions)
Autoflex 3 smartbeam tof tof 200 system, by Bruker (2 mentions)
Gamma 32p atp, by PerkinElmer (2 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Megascript sp6 transcription kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Dig labeling kit, by Roche (1 mentions)
Qiaquick nucleotide removal kit, by Qiagen (1 mentions)
S2 instrument, by Covaris (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Wt ovation pico rna amplification system, by Tecan (1 mentions)
Synapt g2 hdms, by Waters Corporation (1 mentions)
Alexa fluor 488 succinimidyl ester, by Thermo Fisher Scientific (1 mentions)
Spintrap, by Cytiva (1 mentions)
20 protocols using micro bio spin 30 column
1
Radiolabeled RNA Probe for RCNMV RNA1 3' UTR
2
In Situ Hybridization of Cartilage Markers
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In situ hybridization was performed for perichondrium cambium layer markers Lgals1 and cartilage markers Col2a1 and collagen type X alpha 1 Col10a1 in 3‐day‐old rat proximal tibias and in vitro chondrocyte pellets. DNA templates for riboprobe transcription were amplified by PCR from epiphyseal cDNA using custom‐designed primers (Table 1 ) containing a T7 (for sense probes) or Sp6 (for antisense probes) promoter.
PCR products were purified by agarose gel electrophoresis and QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). Single‐stranded riboprobes for in situ hybridization were transcribed using DIG Labeling Kit (Roche Diagnostics, Mannheim, Germany) that incorporates a digoxigenin‐ (DIG‐) conjugated uracil every 20 to 25 nucleotides. Labeled RNA probes were purified using Micro Bio‐Spin 30 Columns (Bio‐Rad, Hercules, CA, USA) and quantified using a NanoDrop Spectrophotometer (Thermo Fisher Scientific).
PCR products were purified by agarose gel electrophoresis and QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). Single‐stranded riboprobes for in situ hybridization were transcribed using DIG Labeling Kit (Roche Diagnostics, Mannheim, Germany) that incorporates a digoxigenin‐ (DIG‐) conjugated uracil every 20 to 25 nucleotides. Labeled RNA probes were purified using Micro Bio‐Spin 30 Columns (Bio‐Rad, Hercules, CA, USA) and quantified using a NanoDrop Spectrophotometer (Thermo Fisher Scientific).
3
Radioactive Probe Synthesis for CRHR1, MR, GR
4
Ribonucleotide Probe Detection of GR and MR mRNA
5
Enzymatic Oxidation of Genomic DNA
6
TAB-Seq and TA-RRBS for Oxidized DNA Analysis
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For TAB-Seq, genomic DNAs and spike-in control DNAs were sheared to an average size of 200 bp using a Covaris S2 instrument. Glycosylation and oxidation of genomic DNAs was performed following a previous protocol with small modifications [44 (link)]. Briefly, 5 μg of sheared genomic DNA or with spike-in controls was initially glycosylated using β-glucosyltransferase proteins that were expressed and purified as previously described. Then, the DNA was purified using the QIAquick Nucleotide Removal Kit (Qiagen). Next, the oxidation reaction was performed using 1.5 μg of glycosylated DNA and 30 μL recombinant mTet1 protein in a 150 μL reaction solution and incubated for 1 h at 37°C. After proteinase K treatment, the oxidized DNA was first purified with Micro Bio-Spin 30 Columns (Bio-Rad) and then with 1.8 × Ampure XP Beads (Beckman) following the manufacturer’s suggestions.
For Tet-assisted reduced representation bisulfite sequencing (TA-RRBS), genomic DNAs extracted from purified neuron nucleus were first digested with MspI for 3 h at 37°C and purified using the QIAquick Nucleotide Removal Kit as described [45 (link)]. Then, glycosylation and oxidation of the DNA was performed similar to the TAB-Seq.
For Tet-assisted reduced representation bisulfite sequencing (TA-RRBS), genomic DNAs extracted from purified neuron nucleus were first digested with MspI for 3 h at 37°C and purified using the QIAquick Nucleotide Removal Kit as described [45 (link)]. Then, glycosylation and oxidation of the DNA was performed similar to the TAB-Seq.
7
Ribonucleotide Probe Preparation
8
RNA Isolation and Amplification for Embryo Samples
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RNA isolation was performed from each sample using a DynabeadsR mRNA Direct™ Micro-Kit (Invitrogen, Grand Island, NY). RNA extraction from a total of 3–4 biological replicates was completed for each developmental stage measured. Each replicate yielded mRNA that was then diluted in 12 µl of ultra-pure water. Due to limited numbers of IVV embryos, cDNA was amplified for selected experiments. All developmental stages for IVV, IVF, SCNT, and parthenogenetic embryos were amplified, as well as oocytes and embryos that were treated with inhibitors after fertilization. Microinjected oocytes and embryos and samples treated with inhibitors during fertilization and culture were not amplified.
To obtain amplified cDNA, 5 µl of the mRNA sample was used to synthesize first- and second-strand cDNA with the WT-Ovation™ Pico RNA Amplification System (NuGEN Technologies, Inc., San Carlos, CA). Amplified cDNA samples were purified by Micro Bio-Spin 30 Columns in RNase-Free Tris (Bio-Rad, Hercules, CA), and stored at −80°C. For the purpose of qPCR, an aliquot of purified amplified cDNA template was diluted to 5 ng/µl in ultra-pure water. Unamplified cDNA was synthesized using the entire 12 µl of diluted mRNA for the SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen). Note that unamplified samples were used to generate the data forFigures 3 and 7 .
To obtain amplified cDNA, 5 µl of the mRNA sample was used to synthesize first- and second-strand cDNA with the WT-Ovation™ Pico RNA Amplification System (NuGEN Technologies, Inc., San Carlos, CA). Amplified cDNA samples were purified by Micro Bio-Spin 30 Columns in RNase-Free Tris (Bio-Rad, Hercules, CA), and stored at −80°C. For the purpose of qPCR, an aliquot of purified amplified cDNA template was diluted to 5 ng/µl in ultra-pure water. Unamplified cDNA was synthesized using the entire 12 µl of diluted mRNA for the SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen). Note that unamplified samples were used to generate the data for
9
Ribonucleotide Probe Synthesis
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The ribonucleotide probe was prepared using a rat c-fos 2116 bp template provided by Dr. Victor Viau (Department Cellular and Physiological Sciences, The University of British Columbia, Canada). Probes were labeled with 35S-UTP (Amersham Biosciences, NJ, USA) using Polymerase T7 and Promega Riboprobe System (Promega Corporation, Madison, WI, USA). All probes were purified using Micro Bio-Spin 30 Columns (Bio-Rad, CA, USA). One molar of DTT was added to prevent oxidation.
10
Ion Mobility Mass Spectrometry of Antibodies
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