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Promega rctp
Rctp, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 15 article reviews
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rctp - by Bioz Stars, 2020-01
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Related Articles

Clone Assay:

Article Title: Expression profiling across many samples via manifold-assisted mRNA processing
Article Snippet: A 362-bp fragment of the bcr–abl gene was cloned into plasmid pTML65(A30 ) ( ). .. RNA molecules with A30 -tails were generated by in vitro transcription of the Nsi I-linearized plasmid in 40 mM Tris–HCl pH 7.9, 6 mM MgCl2 , 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol (DTT), 1.5 U/µl human placental ribonuclease inhibitor (HPRI), 0.5 mM rATP, rGTP, rCTP, 1 U/µl T3 RNA polymerase (Promega, WI) and 2.5 µCi/µl [α-32 P]UTP.

Article Title: Involvement of Calpain-Calpastatin in Cigarette Smoke-Induced Inhibition of Lung Endothelial Nitric Oxide Synthase
Article Snippet: Reagents for the cDNA cloning and expressional vector construction of porcine calpastatin were obtained from Invitrogen (Carlsbad, CA), Clontech (Palo Alto, CA), and ATCC (Manassas, VA), respectively. .. Effectene was obtained from Qiagen (Valencia, CA). rATP, rUTP, rGTP, and rCTP were obtained from Promega (Madison, WI).

Amplification:

Article Title: RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution
Article Snippet: Run off transcription on the linear DNA template was performed by adding 40 mM Tris-HCl (pH 7.9), 6 mM MgCl2 , 2 mM spermidine, 10 mM dithiothreitol, 1 mM each of rCTP, rUTP, rGTP, and rATP, 2 U RNase inhibitor (Promega), 20 U T7 polymerase (Promega) to 200 ng DNA template and incubating at 37°C overnight. .. RNA was DNase-treated to remove the DNA template (RQ1 DNase, Promega) and purified (RNeasy, Qiagen) prior to amplification with Qβ replicase.

Article Title: Sequential RNA degradation pathways provide a fail-safe mechanism to limit the accumulation of unspliced transcripts in Saccharomyces cerevisiae
Article Snippet: Riboprobe generation encompassed utilization of an amplified intron containing the T3 RNA Polymerase promoter at the 3′ end. .. Briefly, 3 μL of product was utilized as a template with 1 μL each of rATP, rCTP, and rGTP, and 2–3 μL of [α-32- P]UTP; 4 μL of 5× Promega transcription buffer; 2 μL of 100 mM DTT; and 1 μL of T3 RNA polymerase (Promega).

Article Title: Defining a novel leptin–melanocortin–kisspeptin pathway involved in the metabolic control of puberty
Article Snippet: First, a DNA template was synthesized by PCR using specific primers for Kiss1 cDNA amplification carrying at their 5′-end sequences for synthetic promoters for bacteriophage-encoded DNA-dependent RNA polymerases (T7 and T3). .. For the generation of the antisense Kiss1 riboprobe, the product of the PCR was used as template for the transcription reaction, as follows (final volume of 20 μl): 250 Ci [33P]-UTP (Perkin Elmer, Massachusetts, USA), 0.5 μg of template, 2 μl 3NTPs (5 mM rATP, rCTP and rGTP), 1 μl RNasin Ribonuclease Inhibitor (Promega), 4 μl transcription buffer, and 2 μl T7 RNA polymerase (Promega).

Synthesized:

Article Title: Involvement of Calpain-Calpastatin in Cigarette Smoke-Induced Inhibition of Lung Endothelial Nitric Oxide Synthase
Article Snippet: Oligodeoxyribonucleotides (ODN) were synthesized by Invitrogen. .. Effectene was obtained from Qiagen (Valencia, CA). rATP, rUTP, rGTP, and rCTP were obtained from Promega (Madison, WI).

Article Title: Functional analysis of a tripartite stability element within the CD40 ligand 3? untranslated region
Article Snippet: The following probes were generated for use in RNA-binding studies using 5′ primers that contained a T7 promoter and unique 3′ primers: T7-1300-E1, 5′-cgtaatacgactcactatagggctagaacgtctaacacagtggaga-3′ (forward) and 5′tgaaagagagagatggagagagagagagagatt-3′ (reverse); T7-1349-E1, 5′-cgtaatacgactcactataggggccaccctctcggacagt-3′ (forward) and 5′-tgaaagagagagatggagagagagagagagatt-3′ (reverse); T7-E5- Hae III and E5- Hin fI templates were synthesized using primers: 5′-cgtaatacgactcactatagggagtctcttccctcccccagtctctctt-3′ (forward) and 5′-agagaactgactagcaacggc-ctga-3′ (reverse), followed by digestion with Hae III and Hin fI enzymes (Promega, Madison, WI) respectively; the T7-E5-1518, 5′-cgtaatacgactcactatagggagtctcttccctcccccagtctctctt-3′ (forward) and 5′ttagaaagggggattga-3′ (reverse); the T7-E5-HΔCa, 5′-cgtaatacgactcactatagggagtctcttccctcccccagtctctctt-3′ (forward) and 5′-cctgactcttagaaagggggattg-3′ (reverse); T7-1518- Hae III probe, 5′-tccccctttccgtaatacgactcactatagggtaacacacacaca-3′ (forward), and 5′-agagaactgactagcaacggcctga-3′ (reverse), followed by digestion with Hae III. .. For the synthesis of 32 P-labelled RNA probes 0·5 μg template DNA; 0·4 m m each of rATP, rGTP and rCTP; 0·04 m m rUTP; 30 m m DTT; 20 U of RNasin (Promega); 2·5 m m cap analogue (Amersham Biosciences, Piscataway, NJ); T7 transcription buffer (40 m m Tris–HCl 7·9; 6 m m MgCl2 ; 2 m m spermidine and 10 m m NaCl); 25–40 μCi [α-32 P]rUTP and 5 U T7 RNA polymerase (Promega) were used at 37° for 1 hr, treated with RQ1 RNase-free DNase at 37° for 15 min and centrifuged through G25 columns (Amersham Biosciences) to remove the unincorporated nucleotides.

Article Title: Defining a novel leptin–melanocortin–kisspeptin pathway involved in the metabolic control of puberty
Article Snippet: First, a DNA template was synthesized by PCR using specific primers for Kiss1 cDNA amplification carrying at their 5′-end sequences for synthetic promoters for bacteriophage-encoded DNA-dependent RNA polymerases (T7 and T3). .. For the generation of the antisense Kiss1 riboprobe, the product of the PCR was used as template for the transcription reaction, as follows (final volume of 20 μl): 250 Ci [33P]-UTP (Perkin Elmer, Massachusetts, USA), 0.5 μg of template, 2 μl 3NTPs (5 mM rATP, rCTP and rGTP), 1 μl RNasin Ribonuclease Inhibitor (Promega), 4 μl transcription buffer, and 2 μl T7 RNA polymerase (Promega).

Article Title: mRNA Decay during Herpes Simplex Virus (HSV) Infections: Mutations That Affect Translation of an mRNA Influence the Sites at Which It Is Cleaved by the HSV Virion Host Shutoff (Vhs) Protein
Article Snippet: .. Capped internally labeled pBK2 mRNA was synthesized using an SP6 Riboprobe in vitro transcription system (Promega) in reactions that included 0.5 mM (each) rATP, rUTP, and rCTP, 50 μM rGTP, 50 μCi [α-32 P]GTP, and 0.5 mM Ribo m7 G cap analog (Promega). .. After in vitro transcription, template DNA was removed by digestion with RQ1 DNase (Promega). mRNAs were extracted twice with phenol-chloroform, precipitated from ethanol, and resuspended in a small volume of water.

Article Title: Chemokine Gene Expression in Astrocytes of Borna Disease Virus-Infected Rats and Mice in the Absence of Inflammation
Article Snippet: .. The radiolabeled antisense RPA probe set was synthesized in a volume of 20 μl containing 100 μCi of [α-32 P]UTP (3,000 Ci/mmol); dithiothreitol (200 nmol); transcription buffer (Promega); 1 μl of template DNA; rUTP (61 pmol); rGTP, rATP, and rCTP (2.75 nmol each); RNase inhibitor (28 U; Pharmacia); and T7 polymerase (20 U; Promega). .. After 1 h of incubation at 37°C, the template DNAs were digested by treatment with DNase I (4 U; Ambion) for 30 min at 37°C.

Construct:

Article Title: Expression profiling across many samples via manifold-assisted mRNA processing
Article Snippet: Paragraph title: Plasmid construct and in vitro transcription ... RNA molecules with A30 -tails were generated by in vitro transcription of the Nsi I-linearized plasmid in 40 mM Tris–HCl pH 7.9, 6 mM MgCl2 , 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol (DTT), 1.5 U/µl human placental ribonuclease inhibitor (HPRI), 0.5 mM rATP, rGTP, rCTP, 1 U/µl T3 RNA polymerase (Promega, WI) and 2.5 µCi/µl [α-32 P]UTP.

Article Title: Stem-Loop IV in the 5? Untranslated Region Is a cis-Acting Element in Bovine Coronavirus Defective Interfering RNA Replication
Article Snippet: For synthesis of uniformly radiolabeled RNA probe, plasmid DNA of wild-type (wt) pSLIV(PB) or the PB modifications of stem-loop IV mutants (Table ) was linearized with NcoI, and 2.5 μg was transcribed in a 50-μl reaction volume with 40 U T7 RNA polymerase (Promega) at 37°C for 1 h in the presence of 120 μCi [α-32 P]UTP (300 Ci/mmole; ICN), 0.5 mM each of rATP, rCTP, and rGTP (Promega), 12 μM UTP (Promega), 10 mM DTT, and 5 mg acetylated bovine serum albumin (Promega). .. Transcripts were 113 nt in length from all constructs except for those from pSLIV-mutdel(PB), which were 93 nt in length.

Electrophoresis:

Article Title: Defining a novel leptin–melanocortin–kisspeptin pathway involved in the metabolic control of puberty
Article Snippet: After electrophoresis on a 2% agarose (w/v) gel, a single DNA fragment was obtained of the expected size and gel purified with a QiaQuick gel extraction kit (Qiagen). .. For the generation of the antisense Kiss1 riboprobe, the product of the PCR was used as template for the transcription reaction, as follows (final volume of 20 μl): 250 Ci [33P]-UTP (Perkin Elmer, Massachusetts, USA), 0.5 μg of template, 2 μl 3NTPs (5 mM rATP, rCTP and rGTP), 1 μl RNasin Ribonuclease Inhibitor (Promega), 4 μl transcription buffer, and 2 μl T7 RNA polymerase (Promega).

Incubation:

Article Title: Sequential RNA degradation pathways provide a fail-safe mechanism to limit the accumulation of unspliced transcripts in Saccharomyces cerevisiae
Article Snippet: Briefly, 3 μL of product was utilized as a template with 1 μL each of rATP, rCTP, and rGTP, and 2–3 μL of [α-32- P]UTP; 4 μL of 5× Promega transcription buffer; 2 μL of 100 mM DTT; and 1 μL of T3 RNA polymerase (Promega). .. The in vitro transcription reaction was incubated at 37°C for 1 h, followed by the addition of 1 μL of DNase (to degrade template DNA), and further incubated at 37°C for 15 min.

Article Title: Expression profiling across many samples via manifold-assisted mRNA processing
Article Snippet: RNA molecules with A30 -tails were generated by in vitro transcription of the Nsi I-linearized plasmid in 40 mM Tris–HCl pH 7.9, 6 mM MgCl2 , 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol (DTT), 1.5 U/µl human placental ribonuclease inhibitor (HPRI), 0.5 mM rATP, rGTP, rCTP, 1 U/µl T3 RNA polymerase (Promega, WI) and 2.5 µCi/µl [α-32 P]UTP. .. The reaction was incubated at 37°C for 60 min, followed by an incubation with 0.5 U/µl RQ DNase I (Promega) for 30 min.

Article Title: A Unique Mitochondrial Transcription Factor B Protein in Dictyostelium discoideum
Article Snippet: The reaction consisted of; 5× transcription buffer (Promega): 4 µL, 100 mM DTT: 2 µL, RNasin (Promega) (40 U): 0.5 µL, 2.5 mM rNTPs (rATP, rGTP and rUTP): 3 µL, 100 µM rCTP: 2.4 µL, [α-32 P] rCTP (10 mCi/mL): 5 µL, linearised pBS-rnl vector: 1–2 µg, T3 RNA polymerase (Promega): 1 µL and DEPC sdH2 O (to 20 µL). .. The reaction was incubated at 37°C for 1 h and the transcribed RNA was precipitated with an equal volume of isopropanol and used for hybridisation.

Article Title: Defining a novel leptin–melanocortin–kisspeptin pathway involved in the metabolic control of puberty
Article Snippet: For the generation of the antisense Kiss1 riboprobe, the product of the PCR was used as template for the transcription reaction, as follows (final volume of 20 μl): 250 Ci [33P]-UTP (Perkin Elmer, Massachusetts, USA), 0.5 μg of template, 2 μl 3NTPs (5 mM rATP, rCTP and rGTP), 1 μl RNasin Ribonuclease Inhibitor (Promega), 4 μl transcription buffer, and 2 μl T7 RNA polymerase (Promega). .. After 120 min of incubation at 37 °C, another 1 μl of T7 RNA was added to the mix, and the reaction was maintained for an additional 60 min at 37 °C.

Article Title: TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus
Article Snippet: For HCV transcripts and use with SP6 polymerase, the mix contained 80 mM HEPES (pH 7.5), 16 mM MgCl2 , 2 mM spermidine, 40 mM DTT, 3.125 mM each of rATP, rCTP and rUTP, 1.5625 mM of rGTP, 1 mM m7 G(5′)ppp(5′)G RNA cap structure analog, 1 U/μl of RNasin, 0.1 μg/μl of plasmid DNA and 0.6 U/μl of SP6 RNA polymerase (Promega). .. After a 2 h incubation at 37°C (HCV) or 40°C (Dengue virus), 0.3 U of T7 RNA polymerase or 0.4 U of SP6 RNA polymerase, respectively, were added per μl of reaction mixture, and the reaction mixture was incubated overnight.

Article Title: Chemokine Gene Expression in Astrocytes of Borna Disease Virus-Infected Rats and Mice in the Absence of Inflammation
Article Snippet: The radiolabeled antisense RPA probe set was synthesized in a volume of 20 μl containing 100 μCi of [α-32 P]UTP (3,000 Ci/mmol); dithiothreitol (200 nmol); transcription buffer (Promega); 1 μl of template DNA; rUTP (61 pmol); rGTP, rATP, and rCTP (2.75 nmol each); RNase inhibitor (28 U; Pharmacia); and T7 polymerase (20 U; Promega). .. After 1 h of incubation at 37°C, the template DNAs were digested by treatment with DNase I (4 U; Ambion) for 30 min at 37°C.

Article Title: Stem-Loop IV in the 5? Untranslated Region Is a cis-Acting Element in Bovine Coronavirus Defective Interfering RNA Replication
Article Snippet: AT 6 hpi, cells were washed with ice cold PBS, scraped into 500 μl cold PBS, pelleted at 600× g at 4°C for 4 min, resuspended in 400 μl lysis buffer (10 mM HEPES [pH 7.5], 3 mM MgCl2 , 14 mM KCl, 5% glycerol [vol/vol], 1.0% Nonidet P-40 [vol/vol], 1 mM dithiothreitol [DTT], 0.1 mM phenylmethylsulfonyl fluoride [PMSF]), incubated 20 min on ice, and then homogenized with 30 strokes in a tight-fitting Dounce homogenizer. .. For synthesis of uniformly radiolabeled RNA probe, plasmid DNA of wild-type (wt) pSLIV(PB) or the PB modifications of stem-loop IV mutants (Table ) was linearized with NcoI, and 2.5 μg was transcribed in a 50-μl reaction volume with 40 U T7 RNA polymerase (Promega) at 37°C for 1 h in the presence of 120 μCi [α-32 P]UTP (300 Ci/mmole; ICN), 0.5 mM each of rATP, rCTP, and rGTP (Promega), 12 μM UTP (Promega), 10 mM DTT, and 5 mg acetylated bovine serum albumin (Promega).

Expressing:

Article Title: Chemokine Gene Expression in Astrocytes of Borna Disease Virus-Infected Rats and Mice in the Absence of Inflammation
Article Snippet: L32 was included in the riboprobe set to detect transcripts of the ribosomal protein L32 (encoded by a housekeeping gene), permitting normalization of chemokine mRNA expression. .. The radiolabeled antisense RPA probe set was synthesized in a volume of 20 μl containing 100 μCi of [α-32 P]UTP (3,000 Ci/mmol); dithiothreitol (200 nmol); transcription buffer (Promega); 1 μl of template DNA; rUTP (61 pmol); rGTP, rATP, and rCTP (2.75 nmol each); RNase inhibitor (28 U; Pharmacia); and T7 polymerase (20 U; Promega).

RNA Binding Assay:

Article Title: Functional analysis of a tripartite stability element within the CD40 ligand 3? untranslated region
Article Snippet: The following probes were generated for use in RNA-binding studies using 5′ primers that contained a T7 promoter and unique 3′ primers: T7-1300-E1, 5′-cgtaatacgactcactatagggctagaacgtctaacacagtggaga-3′ (forward) and 5′tgaaagagagagatggagagagagagagagatt-3′ (reverse); T7-1349-E1, 5′-cgtaatacgactcactataggggccaccctctcggacagt-3′ (forward) and 5′-tgaaagagagagatggagagagagagagagatt-3′ (reverse); T7-E5- Hae III and E5- Hin fI templates were synthesized using primers: 5′-cgtaatacgactcactatagggagtctcttccctcccccagtctctctt-3′ (forward) and 5′-agagaactgactagcaacggc-ctga-3′ (reverse), followed by digestion with Hae III and Hin fI enzymes (Promega, Madison, WI) respectively; the T7-E5-1518, 5′-cgtaatacgactcactatagggagtctcttccctcccccagtctctctt-3′ (forward) and 5′ttagaaagggggattga-3′ (reverse); the T7-E5-HΔCa, 5′-cgtaatacgactcactatagggagtctcttccctcccccagtctctctt-3′ (forward) and 5′-cctgactcttagaaagggggattg-3′ (reverse); T7-1518- Hae III probe, 5′-tccccctttccgtaatacgactcactatagggtaacacacacaca-3′ (forward), and 5′-agagaactgactagcaacggcctga-3′ (reverse), followed by digestion with Hae III. .. For the synthesis of 32 P-labelled RNA probes 0·5 μg template DNA; 0·4 m m each of rATP, rGTP and rCTP; 0·04 m m rUTP; 30 m m DTT; 20 U of RNasin (Promega); 2·5 m m cap analogue (Amersham Biosciences, Piscataway, NJ); T7 transcription buffer (40 m m Tris–HCl 7·9; 6 m m MgCl2 ; 2 m m spermidine and 10 m m NaCl); 25–40 μCi [α-32 P]rUTP and 5 U T7 RNA polymerase (Promega) were used at 37° for 1 hr, treated with RQ1 RNase-free DNase at 37° for 15 min and centrifuged through G25 columns (Amersham Biosciences) to remove the unincorporated nucleotides.

Recombinase Polymerase Amplification:

Article Title: Chemokine Gene Expression in Astrocytes of Borna Disease Virus-Infected Rats and Mice in the Absence of Inflammation
Article Snippet: .. The radiolabeled antisense RPA probe set was synthesized in a volume of 20 μl containing 100 μCi of [α-32 P]UTP (3,000 Ci/mmol); dithiothreitol (200 nmol); transcription buffer (Promega); 1 μl of template DNA; rUTP (61 pmol); rGTP, rATP, and rCTP (2.75 nmol each); RNase inhibitor (28 U; Pharmacia); and T7 polymerase (20 U; Promega). .. After 1 h of incubation at 37°C, the template DNAs were digested by treatment with DNase I (4 U; Ambion) for 30 min at 37°C.

Translocation Assay:

Article Title: Mammalian SRP receptor switches the Sec61 translocase from Sec62 to SRP-dependent translocation
Article Snippet: Paragraph title: In vitro translocation assays ... Transcription reactions were carried out in a volume of 100 μl for 2 h at 37 °C, in the presence of 1 × transcription-optimised buffer (Promega), 10 mM DTT, 0.5 μg of template DNA, 0.25 mM each of rATP, rCTP, rGTP and rUTP (Promega), 0.5 mM Cap analogue (m7 G[5′]ppp[5′]G; New England Biolabs), 80 units of T7 RNA Polymerase (Promega) and 100 units of RNasin Plus RNase Inhibitor (Promega).

Hybridization:

Article Title: Sequential RNA degradation pathways provide a fail-safe mechanism to limit the accumulation of unspliced transcripts in Saccharomyces cerevisiae
Article Snippet: Briefly, 3 μL of product was utilized as a template with 1 μL each of rATP, rCTP, and rGTP, and 2–3 μL of [α-32- P]UTP; 4 μL of 5× Promega transcription buffer; 2 μL of 100 mM DTT; and 1 μL of T3 RNA polymerase (Promega). .. The probe was then added to Church hybridization buffer.

Article Title: A Unique Mitochondrial Transcription Factor B Protein in Dictyostelium discoideum
Article Snippet: The reaction consisted of; 5× transcription buffer (Promega): 4 µL, 100 mM DTT: 2 µL, RNasin (Promega) (40 U): 0.5 µL, 2.5 mM rNTPs (rATP, rGTP and rUTP): 3 µL, 100 µM rCTP: 2.4 µL, [α-32 P] rCTP (10 mCi/mL): 5 µL, linearised pBS-rnl vector: 1–2 µg, T3 RNA polymerase (Promega): 1 µL and DEPC sdH2 O (to 20 µL). .. The reaction was incubated at 37°C for 1 h and the transcribed RNA was precipitated with an equal volume of isopropanol and used for hybridisation.

Article Title: Chemokine Gene Expression in Astrocytes of Borna Disease Virus-Infected Rats and Mice in the Absence of Inflammation
Article Snippet: The radiolabeled antisense RPA probe set was synthesized in a volume of 20 μl containing 100 μCi of [α-32 P]UTP (3,000 Ci/mmol); dithiothreitol (200 nmol); transcription buffer (Promega); 1 μl of template DNA; rUTP (61 pmol); rGTP, rATP, and rCTP (2.75 nmol each); RNase inhibitor (28 U; Pharmacia); and T7 polymerase (20 U; Promega). .. After extraction with phenol-chloroform, probes were precipitated with ethanol in the presence of mussel glycogen (20 μg; Roche Molecular Biochemicals), dried, and dissolved (3 × 105 cpm/μl) in hybridization buffer 1 [40 mM piperazine- N , N ′-bis(2-ethanesulfonic acid) (PIPES) [pH 6.4], 0.4 M NaCl, 1 mM EDTA, 80% formamide].

Countercurrent Chromatography:

Article Title: RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution
Article Snippet: T7 polymerase often inserts a terminal A nucleotide to the 3' CCC sequence, however, this did not appear to significantly influence RNA template recognition. .. Run off transcription on the linear DNA template was performed by adding 40 mM Tris-HCl (pH 7.9), 6 mM MgCl2 , 2 mM spermidine, 10 mM dithiothreitol, 1 mM each of rCTP, rUTP, rGTP, and rATP, 2 U RNase inhibitor (Promega), 20 U T7 polymerase (Promega) to 200 ng DNA template and incubating at 37°C overnight.

Infection:

Article Title: Stem-Loop IV in the 5? Untranslated Region Is a cis-Acting Element in Bovine Coronavirus Defective Interfering RNA Replication
Article Snippet: Briefly, cells at 80% confluence in a 35-mm dish (∼2 × 106 cells yielding lysate for about four gel-shift reactions) were mock infected or infected with BCoV (MOI = 10). .. For synthesis of uniformly radiolabeled RNA probe, plasmid DNA of wild-type (wt) pSLIV(PB) or the PB modifications of stem-loop IV mutants (Table ) was linearized with NcoI, and 2.5 μg was transcribed in a 50-μl reaction volume with 40 U T7 RNA polymerase (Promega) at 37°C for 1 h in the presence of 120 μCi [α-32 P]UTP (300 Ci/mmole; ICN), 0.5 mM each of rATP, rCTP, and rGTP (Promega), 12 μM UTP (Promega), 10 mM DTT, and 5 mg acetylated bovine serum albumin (Promega).

Generated:

Article Title: Sequential RNA degradation pathways provide a fail-safe mechanism to limit the accumulation of unspliced transcripts in Saccharomyces cerevisiae
Article Snippet: With the exception of the SCR1 probe (which was generated through random priming), all probes were antisense intronic riboprobes that spanned the intronic region of each of the target intron-containing genes that were studied. .. Briefly, 3 μL of product was utilized as a template with 1 μL each of rATP, rCTP, and rGTP, and 2–3 μL of [α-32- P]UTP; 4 μL of 5× Promega transcription buffer; 2 μL of 100 mM DTT; and 1 μL of T3 RNA polymerase (Promega).

Article Title: Expression profiling across many samples via manifold-assisted mRNA processing
Article Snippet: .. RNA molecules with A30 -tails were generated by in vitro transcription of the Nsi I-linearized plasmid in 40 mM Tris–HCl pH 7.9, 6 mM MgCl2 , 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol (DTT), 1.5 U/µl human placental ribonuclease inhibitor (HPRI), 0.5 mM rATP, rGTP, rCTP, 1 U/µl T3 RNA polymerase (Promega, WI) and 2.5 µCi/µl [α-32 P]UTP. .. The reaction was incubated at 37°C for 60 min, followed by an incubation with 0.5 U/µl RQ DNase I (Promega) for 30 min.

Article Title: Functional analysis of a tripartite stability element within the CD40 ligand 3? untranslated region
Article Snippet: The following probes were generated for use in RNA-binding studies using 5′ primers that contained a T7 promoter and unique 3′ primers: T7-1300-E1, 5′-cgtaatacgactcactatagggctagaacgtctaacacagtggaga-3′ (forward) and 5′tgaaagagagagatggagagagagagagagatt-3′ (reverse); T7-1349-E1, 5′-cgtaatacgactcactataggggccaccctctcggacagt-3′ (forward) and 5′-tgaaagagagagatggagagagagagagagatt-3′ (reverse); T7-E5- Hae III and E5- Hin fI templates were synthesized using primers: 5′-cgtaatacgactcactatagggagtctcttccctcccccagtctctctt-3′ (forward) and 5′-agagaactgactagcaacggc-ctga-3′ (reverse), followed by digestion with Hae III and Hin fI enzymes (Promega, Madison, WI) respectively; the T7-E5-1518, 5′-cgtaatacgactcactatagggagtctcttccctcccccagtctctctt-3′ (forward) and 5′ttagaaagggggattga-3′ (reverse); the T7-E5-HΔCa, 5′-cgtaatacgactcactatagggagtctcttccctcccccagtctctctt-3′ (forward) and 5′-cctgactcttagaaagggggattg-3′ (reverse); T7-1518- Hae III probe, 5′-tccccctttccgtaatacgactcactatagggtaacacacacaca-3′ (forward), and 5′-agagaactgactagcaacggcctga-3′ (reverse), followed by digestion with Hae III. .. For the synthesis of 32 P-labelled RNA probes 0·5 μg template DNA; 0·4 m m each of rATP, rGTP and rCTP; 0·04 m m rUTP; 30 m m DTT; 20 U of RNasin (Promega); 2·5 m m cap analogue (Amersham Biosciences, Piscataway, NJ); T7 transcription buffer (40 m m Tris–HCl 7·9; 6 m m MgCl2 ; 2 m m spermidine and 10 m m NaCl); 25–40 μCi [α-32 P]rUTP and 5 U T7 RNA polymerase (Promega) were used at 37° for 1 hr, treated with RQ1 RNase-free DNase at 37° for 15 min and centrifuged through G25 columns (Amersham Biosciences) to remove the unincorporated nucleotides.

Article Title: Mammalian SRP receptor switches the Sec61 translocase from Sec62 to SRP-dependent translocation
Article Snippet: In vitro translocation assays Templates for preprolactin , opsin-tagged preprocecropin A, apelin, statherin and cytochrome B5 were generated by PCR . .. Transcription reactions were carried out in a volume of 100 μl for 2 h at 37 °C, in the presence of 1 × transcription-optimised buffer (Promega), 10 mM DTT, 0.5 μg of template DNA, 0.25 mM each of rATP, rCTP, rGTP and rUTP (Promega), 0.5 mM Cap analogue (m7 G[5′]ppp[5′]G; New England Biolabs), 80 units of T7 RNA Polymerase (Promega) and 100 units of RNasin Plus RNase Inhibitor (Promega).

Inhibition:

Article Title: TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus
Article Snippet: Paragraph title: HCV inhibition experiments ... For HCV transcripts and use with SP6 polymerase, the mix contained 80 mM HEPES (pH 7.5), 16 mM MgCl2 , 2 mM spermidine, 40 mM DTT, 3.125 mM each of rATP, rCTP and rUTP, 1.5625 mM of rGTP, 1 mM m7 G(5′)ppp(5′)G RNA cap structure analog, 1 U/μl of RNasin, 0.1 μg/μl of plasmid DNA and 0.6 U/μl of SP6 RNA polymerase (Promega).

Sequencing:

Article Title: RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution
Article Snippet: T7 polymerase often inserts a terminal A nucleotide to the 3' CCC sequence, however, this did not appear to significantly influence RNA template recognition. .. Run off transcription on the linear DNA template was performed by adding 40 mM Tris-HCl (pH 7.9), 6 mM MgCl2 , 2 mM spermidine, 10 mM dithiothreitol, 1 mM each of rCTP, rUTP, rGTP, and rATP, 2 U RNase inhibitor (Promega), 20 U T7 polymerase (Promega) to 200 ng DNA template and incubating at 37°C overnight.

Labeling:

Article Title: Defining a novel leptin–melanocortin–kisspeptin pathway involved in the metabolic control of puberty
Article Snippet: For the generation of the antisense Kiss1 riboprobe, the product of the PCR was used as template for the transcription reaction, as follows (final volume of 20 μl): 250 Ci [33P]-UTP (Perkin Elmer, Massachusetts, USA), 0.5 μg of template, 2 μl 3NTPs (5 mM rATP, rCTP and rGTP), 1 μl RNasin Ribonuclease Inhibitor (Promega), 4 μl transcription buffer, and 2 μl T7 RNA polymerase (Promega). .. DEPC water was added to a final volume of 50 μl, and the labeled riboprobe was purified using illustra ProbeQuant G-50 Micro Columns (GE Healthcare, UK).

Article Title: mRNA Decay during Herpes Simplex Virus (HSV) Infections: Mutations That Affect Translation of an mRNA Influence the Sites at Which It Is Cleaved by the HSV Virion Host Shutoff (Vhs) Protein
Article Snippet: .. Capped internally labeled pBK2 mRNA was synthesized using an SP6 Riboprobe in vitro transcription system (Promega) in reactions that included 0.5 mM (each) rATP, rUTP, and rCTP, 50 μM rGTP, 50 μCi [α-32 P]GTP, and 0.5 mM Ribo m7 G cap analog (Promega). .. After in vitro transcription, template DNA was removed by digestion with RQ1 DNase (Promega). mRNAs were extracted twice with phenol-chloroform, precipitated from ethanol, and resuspended in a small volume of water.

Purification:

Article Title: RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution
Article Snippet: Run off transcription on the linear DNA template was performed by adding 40 mM Tris-HCl (pH 7.9), 6 mM MgCl2 , 2 mM spermidine, 10 mM dithiothreitol, 1 mM each of rCTP, rUTP, rGTP, and rATP, 2 U RNase inhibitor (Promega), 20 U T7 polymerase (Promega) to 200 ng DNA template and incubating at 37°C overnight. .. RNA was DNase-treated to remove the DNA template (RQ1 DNase, Promega) and purified (RNeasy, Qiagen) prior to amplification with Qβ replicase.

Article Title: RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution
Article Snippet: The mRNA was mixed with 40 mM Tris-HCl (pH 7.9), 21 mM MgCl2 , 2 mM spermidine, 10 mM dithiothreitol, 1 mM each of rCTP, rUTP, rGTP, and rATP, 2 U RNase inhibitor (Promega) and 200 nM Qβ replicase (prepared in house following the method of Moody et al . .. The remaining mRNA was purified (RNeasy; Qiagen) to remove excess MgCl2 prior to ribosome display.

Article Title: Mammalian SRP receptor switches the Sec61 translocase from Sec62 to SRP-dependent translocation
Article Snippet: Transcription reactions were carried out in a volume of 100 μl for 2 h at 37 °C, in the presence of 1 × transcription-optimised buffer (Promega), 10 mM DTT, 0.5 μg of template DNA, 0.25 mM each of rATP, rCTP, rGTP and rUTP (Promega), 0.5 mM Cap analogue (m7 G[5′]ppp[5′]G; New England Biolabs), 80 units of T7 RNA Polymerase (Promega) and 100 units of RNasin Plus RNase Inhibitor (Promega). .. Transcripts were then purified with an RNeasy Mini kit (Qiagen) according to the manufacturer's instructions.

Article Title: Defining a novel leptin–melanocortin–kisspeptin pathway involved in the metabolic control of puberty
Article Snippet: After electrophoresis on a 2% agarose (w/v) gel, a single DNA fragment was obtained of the expected size and gel purified with a QiaQuick gel extraction kit (Qiagen). .. For the generation of the antisense Kiss1 riboprobe, the product of the PCR was used as template for the transcription reaction, as follows (final volume of 20 μl): 250 Ci [33P]-UTP (Perkin Elmer, Massachusetts, USA), 0.5 μg of template, 2 μl 3NTPs (5 mM rATP, rCTP and rGTP), 1 μl RNasin Ribonuclease Inhibitor (Promega), 4 μl transcription buffer, and 2 μl T7 RNA polymerase (Promega).

Article Title: TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus
Article Snippet: DNA was purified by using the NucleoSpin Gel and PCR Clean up Kit (Macherey Nagel, Düren, Germany) according to the manufacturer's instructions. .. For HCV transcripts and use with SP6 polymerase, the mix contained 80 mM HEPES (pH 7.5), 16 mM MgCl2 , 2 mM spermidine, 40 mM DTT, 3.125 mM each of rATP, rCTP and rUTP, 1.5625 mM of rGTP, 1 mM m7 G(5′)ppp(5′)G RNA cap structure analog, 1 U/μl of RNasin, 0.1 μg/μl of plasmid DNA and 0.6 U/μl of SP6 RNA polymerase (Promega).

Article Title: Chemokine Gene Expression in Astrocytes of Borna Disease Virus-Infected Rats and Mice in the Absence of Inflammation
Article Snippet: Purified linearized DNAs were pooled at a concentration of 50 ng each per μl. .. The radiolabeled antisense RPA probe set was synthesized in a volume of 20 μl containing 100 μCi of [α-32 P]UTP (3,000 Ci/mmol); dithiothreitol (200 nmol); transcription buffer (Promega); 1 μl of template DNA; rUTP (61 pmol); rGTP, rATP, and rCTP (2.75 nmol each); RNase inhibitor (28 U; Pharmacia); and T7 polymerase (20 U; Promega).

Polymerase Chain Reaction:

Article Title: Functional analysis of a tripartite stability element within the CD40 ligand 3? untranslated region
Article Snippet: Polymerase chain reaction (PCR) was carried out in 10 m m Tris–HCl (pH 9·0), 50 m m KCl, 0·1%Triton X-100, 1·5 m m MgCl2 , 0·2 m m dNTPs, 200 ng of each primer, 50 ng of DNA template, and 2·5 U Taq polymerase (Promega). .. For the synthesis of 32 P-labelled RNA probes 0·5 μg template DNA; 0·4 m m each of rATP, rGTP and rCTP; 0·04 m m rUTP; 30 m m DTT; 20 U of RNasin (Promega); 2·5 m m cap analogue (Amersham Biosciences, Piscataway, NJ); T7 transcription buffer (40 m m Tris–HCl 7·9; 6 m m MgCl2 ; 2 m m spermidine and 10 m m NaCl); 25–40 μCi [α-32 P]rUTP and 5 U T7 RNA polymerase (Promega) were used at 37° for 1 hr, treated with RQ1 RNase-free DNase at 37° for 15 min and centrifuged through G25 columns (Amersham Biosciences) to remove the unincorporated nucleotides.

Article Title: Mammalian SRP receptor switches the Sec61 translocase from Sec62 to SRP-dependent translocation
Article Snippet: In vitro translocation assays Templates for preprolactin , opsin-tagged preprocecropin A, apelin, statherin and cytochrome B5 were generated by PCR . .. Transcription reactions were carried out in a volume of 100 μl for 2 h at 37 °C, in the presence of 1 × transcription-optimised buffer (Promega), 10 mM DTT, 0.5 μg of template DNA, 0.25 mM each of rATP, rCTP, rGTP and rUTP (Promega), 0.5 mM Cap analogue (m7 G[5′]ppp[5′]G; New England Biolabs), 80 units of T7 RNA Polymerase (Promega) and 100 units of RNasin Plus RNase Inhibitor (Promega).

Article Title: Defining a novel leptin–melanocortin–kisspeptin pathway involved in the metabolic control of puberty
Article Snippet: .. For the generation of the antisense Kiss1 riboprobe, the product of the PCR was used as template for the transcription reaction, as follows (final volume of 20 μl): 250 Ci [33P]-UTP (Perkin Elmer, Massachusetts, USA), 0.5 μg of template, 2 μl 3NTPs (5 mM rATP, rCTP and rGTP), 1 μl RNasin Ribonuclease Inhibitor (Promega), 4 μl transcription buffer, and 2 μl T7 RNA polymerase (Promega). .. After 120 min of incubation at 37 °C, another 1 μl of T7 RNA was added to the mix, and the reaction was maintained for an additional 60 min at 37 °C.

Article Title: TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus
Article Snippet: DNA was purified by using the NucleoSpin Gel and PCR Clean up Kit (Macherey Nagel, Düren, Germany) according to the manufacturer's instructions. .. For HCV transcripts and use with SP6 polymerase, the mix contained 80 mM HEPES (pH 7.5), 16 mM MgCl2 , 2 mM spermidine, 40 mM DTT, 3.125 mM each of rATP, rCTP and rUTP, 1.5625 mM of rGTP, 1 mM m7 G(5′)ppp(5′)G RNA cap structure analog, 1 U/μl of RNasin, 0.1 μg/μl of plasmid DNA and 0.6 U/μl of SP6 RNA polymerase (Promega).

Electrophoretic Mobility Shift Assay:

Article Title: Stem-Loop IV in the 5? Untranslated Region Is a cis-Acting Element in Bovine Coronavirus Defective Interfering RNA Replication
Article Snippet: Briefly, cells at 80% confluence in a 35-mm dish (∼2 × 106 cells yielding lysate for about four gel-shift reactions) were mock infected or infected with BCoV (MOI = 10). .. For synthesis of uniformly radiolabeled RNA probe, plasmid DNA of wild-type (wt) pSLIV(PB) or the PB modifications of stem-loop IV mutants (Table ) was linearized with NcoI, and 2.5 μg was transcribed in a 50-μl reaction volume with 40 U T7 RNA polymerase (Promega) at 37°C for 1 h in the presence of 120 μCi [α-32 P]UTP (300 Ci/mmole; ICN), 0.5 mM each of rATP, rCTP, and rGTP (Promega), 12 μM UTP (Promega), 10 mM DTT, and 5 mg acetylated bovine serum albumin (Promega).

Gel Extraction:

Article Title: Defining a novel leptin–melanocortin–kisspeptin pathway involved in the metabolic control of puberty
Article Snippet: After electrophoresis on a 2% agarose (w/v) gel, a single DNA fragment was obtained of the expected size and gel purified with a QiaQuick gel extraction kit (Qiagen). .. For the generation of the antisense Kiss1 riboprobe, the product of the PCR was used as template for the transcription reaction, as follows (final volume of 20 μl): 250 Ci [33P]-UTP (Perkin Elmer, Massachusetts, USA), 0.5 μg of template, 2 μl 3NTPs (5 mM rATP, rCTP and rGTP), 1 μl RNasin Ribonuclease Inhibitor (Promega), 4 μl transcription buffer, and 2 μl T7 RNA polymerase (Promega).

Plasmid Preparation:

Article Title: Expression profiling across many samples via manifold-assisted mRNA processing
Article Snippet: .. RNA molecules with A30 -tails were generated by in vitro transcription of the Nsi I-linearized plasmid in 40 mM Tris–HCl pH 7.9, 6 mM MgCl2 , 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol (DTT), 1.5 U/µl human placental ribonuclease inhibitor (HPRI), 0.5 mM rATP, rGTP, rCTP, 1 U/µl T3 RNA polymerase (Promega, WI) and 2.5 µCi/µl [α-32 P]UTP. .. The reaction was incubated at 37°C for 60 min, followed by an incubation with 0.5 U/µl RQ DNase I (Promega) for 30 min.

Article Title: Involvement of Calpain-Calpastatin in Cigarette Smoke-Induced Inhibition of Lung Endothelial Nitric Oxide Synthase
Article Snippet: Reagents for the cDNA cloning and expressional vector construction of porcine calpastatin were obtained from Invitrogen (Carlsbad, CA), Clontech (Palo Alto, CA), and ATCC (Manassas, VA), respectively. .. Effectene was obtained from Qiagen (Valencia, CA). rATP, rUTP, rGTP, and rCTP were obtained from Promega (Madison, WI).

Article Title: A Unique Mitochondrial Transcription Factor B Protein in Dictyostelium discoideum
Article Snippet: .. The reaction consisted of; 5× transcription buffer (Promega): 4 µL, 100 mM DTT: 2 µL, RNasin (Promega) (40 U): 0.5 µL, 2.5 mM rNTPs (rATP, rGTP and rUTP): 3 µL, 100 µM rCTP: 2.4 µL, [α-32 P] rCTP (10 mCi/mL): 5 µL, linearised pBS-rnl vector: 1–2 µg, T3 RNA polymerase (Promega): 1 µL and DEPC sdH2 O (to 20 µL). .. The reaction was incubated at 37°C for 1 h and the transcribed RNA was precipitated with an equal volume of isopropanol and used for hybridisation.

Article Title: TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus
Article Snippet: .. For HCV transcripts and use with SP6 polymerase, the mix contained 80 mM HEPES (pH 7.5), 16 mM MgCl2 , 2 mM spermidine, 40 mM DTT, 3.125 mM each of rATP, rCTP and rUTP, 1.5625 mM of rGTP, 1 mM m7 G(5′)ppp(5′)G RNA cap structure analog, 1 U/μl of RNasin, 0.1 μg/μl of plasmid DNA and 0.6 U/μl of SP6 RNA polymerase (Promega). .. After a 2 h incubation at 37°C (HCV) or 40°C (Dengue virus), 0.3 U of T7 RNA polymerase or 0.4 U of SP6 RNA polymerase, respectively, were added per μl of reaction mixture, and the reaction mixture was incubated overnight.

Article Title: mRNA Decay during Herpes Simplex Virus (HSV) Infections: Mutations That Affect Translation of an mRNA Influence the Sites at Which It Is Cleaved by the HSV Virion Host Shutoff (Vhs) Protein
Article Snippet: Capped internally labeled pBK2 mRNA was synthesized using an SP6 Riboprobe in vitro transcription system (Promega) in reactions that included 0.5 mM (each) rATP, rUTP, and rCTP, 50 μM rGTP, 50 μCi [α-32 P]GTP, and 0.5 mM Ribo m7 G cap analog (Promega). .. To produce pBK2 mRNA with a 32 P-labeled cap and a cold poly(A) tail, uncapped unlabeled pBK2 mRNA with a 35-nucleotide poly(A) tail encoded by the plasmid was synthesized using an SP6 RiboMAX in vitro transcription kit (Promega).

Article Title: Stem-Loop IV in the 5? Untranslated Region Is a cis-Acting Element in Bovine Coronavirus Defective Interfering RNA Replication
Article Snippet: .. For synthesis of uniformly radiolabeled RNA probe, plasmid DNA of wild-type (wt) pSLIV(PB) or the PB modifications of stem-loop IV mutants (Table ) was linearized with NcoI, and 2.5 μg was transcribed in a 50-μl reaction volume with 40 U T7 RNA polymerase (Promega) at 37°C for 1 h in the presence of 120 μCi [α-32 P]UTP (300 Ci/mmole; ICN), 0.5 mM each of rATP, rCTP, and rGTP (Promega), 12 μM UTP (Promega), 10 mM DTT, and 5 mg acetylated bovine serum albumin (Promega). .. Radiolabeled RNA was treated with 2.5 U RNase-free DNase (Promega) at 37°C for 30 min, phenol-chloroform extracted, electrophoretically resolved on an 8 M urea-6% polyacrylamide gel after the addition of 50 μl loading dye (95% formamide, 20 mM EDTA, 0.3% bromophenol blue and xylene cyanol [wt/vol]).

In Vitro:

Article Title: Sequential RNA degradation pathways provide a fail-safe mechanism to limit the accumulation of unspliced transcripts in Saccharomyces cerevisiae
Article Snippet: Briefly, 3 μL of product was utilized as a template with 1 μL each of rATP, rCTP, and rGTP, and 2–3 μL of [α-32- P]UTP; 4 μL of 5× Promega transcription buffer; 2 μL of 100 mM DTT; and 1 μL of T3 RNA polymerase (Promega). .. The in vitro transcription reaction was incubated at 37°C for 1 h, followed by the addition of 1 μL of DNase (to degrade template DNA), and further incubated at 37°C for 15 min.

Article Title: Expression profiling across many samples via manifold-assisted mRNA processing
Article Snippet: .. RNA molecules with A30 -tails were generated by in vitro transcription of the Nsi I-linearized plasmid in 40 mM Tris–HCl pH 7.9, 6 mM MgCl2 , 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol (DTT), 1.5 U/µl human placental ribonuclease inhibitor (HPRI), 0.5 mM rATP, rGTP, rCTP, 1 U/µl T3 RNA polymerase (Promega, WI) and 2.5 µCi/µl [α-32 P]UTP. .. The reaction was incubated at 37°C for 60 min, followed by an incubation with 0.5 U/µl RQ DNase I (Promega) for 30 min.

Article Title: A Unique Mitochondrial Transcription Factor B Protein in Dictyostelium discoideum
Article Snippet: Preparation of Radiolabelled RNA Probes Synthesis of gene-specific radiolabelled RNA probes was performed via in vitro transcription of RNA using a pBS-rnl vector to create an antisense rnl RNA probe. .. The reaction consisted of; 5× transcription buffer (Promega): 4 µL, 100 mM DTT: 2 µL, RNasin (Promega) (40 U): 0.5 µL, 2.5 mM rNTPs (rATP, rGTP and rUTP): 3 µL, 100 µM rCTP: 2.4 µL, [α-32 P] rCTP (10 mCi/mL): 5 µL, linearised pBS-rnl vector: 1–2 µg, T3 RNA polymerase (Promega): 1 µL and DEPC sdH2 O (to 20 µL).

Article Title: Mammalian SRP receptor switches the Sec61 translocase from Sec62 to SRP-dependent translocation
Article Snippet: Paragraph title: In vitro translocation assays ... Transcription reactions were carried out in a volume of 100 μl for 2 h at 37 °C, in the presence of 1 × transcription-optimised buffer (Promega), 10 mM DTT, 0.5 μg of template DNA, 0.25 mM each of rATP, rCTP, rGTP and rUTP (Promega), 0.5 mM Cap analogue (m7 G[5′]ppp[5′]G; New England Biolabs), 80 units of T7 RNA Polymerase (Promega) and 100 units of RNasin Plus RNase Inhibitor (Promega).

Article Title: TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus
Article Snippet: In vitro transcription reaction mixtures (total volume 100 μl) for use with T7 polymerase contained 80 mM HEPES (pH 7.5), 12 mM MgCl2 , 2 mM spermidine, 40 mM dithiothreitol (DTT), 3.125 mM of each nucleoside triphosphate, 1 U/μl RNasin (Promega), 0.1 μg/μl of plasmid DNA and 0.6 U/μl of T7 RNA polymerase (Promega). .. For HCV transcripts and use with SP6 polymerase, the mix contained 80 mM HEPES (pH 7.5), 16 mM MgCl2 , 2 mM spermidine, 40 mM DTT, 3.125 mM each of rATP, rCTP and rUTP, 1.5625 mM of rGTP, 1 mM m7 G(5′)ppp(5′)G RNA cap structure analog, 1 U/μl of RNasin, 0.1 μg/μl of plasmid DNA and 0.6 U/μl of SP6 RNA polymerase (Promega).

Article Title: mRNA Decay during Herpes Simplex Virus (HSV) Infections: Mutations That Affect Translation of an mRNA Influence the Sites at Which It Is Cleaved by the HSV Virion Host Shutoff (Vhs) Protein
Article Snippet: .. Capped internally labeled pBK2 mRNA was synthesized using an SP6 Riboprobe in vitro transcription system (Promega) in reactions that included 0.5 mM (each) rATP, rUTP, and rCTP, 50 μM rGTP, 50 μCi [α-32 P]GTP, and 0.5 mM Ribo m7 G cap analog (Promega). .. After in vitro transcription, template DNA was removed by digestion with RQ1 DNase (Promega). mRNAs were extracted twice with phenol-chloroform, precipitated from ethanol, and resuspended in a small volume of water.

Protein Binding:

Article Title: Stem-Loop IV in the 5? Untranslated Region Is a cis-Acting Element in Bovine Coronavirus Defective Interfering RNA Replication
Article Snippet: Paragraph title: Protein binding assays. ... For synthesis of uniformly radiolabeled RNA probe, plasmid DNA of wild-type (wt) pSLIV(PB) or the PB modifications of stem-loop IV mutants (Table ) was linearized with NcoI, and 2.5 μg was transcribed in a 50-μl reaction volume with 40 U T7 RNA polymerase (Promega) at 37°C for 1 h in the presence of 120 μCi [α-32 P]UTP (300 Ci/mmole; ICN), 0.5 mM each of rATP, rCTP, and rGTP (Promega), 12 μM UTP (Promega), 10 mM DTT, and 5 mg acetylated bovine serum albumin (Promega).

Produced:

Article Title: mRNA Decay during Herpes Simplex Virus (HSV) Infections: Mutations That Affect Translation of an mRNA Influence the Sites at Which It Is Cleaved by the HSV Virion Host Shutoff (Vhs) Protein
Article Snippet: Target mRNAs for in vitro mRNA degradation assays were produced by in vitro transcription of EcoRI-linearized pBK2 or SpeI-linearized pCITE-RLuc using SP6 or T7 RNA polymerase, respectively. .. Capped internally labeled pBK2 mRNA was synthesized using an SP6 Riboprobe in vitro transcription system (Promega) in reactions that included 0.5 mM (each) rATP, rUTP, and rCTP, 50 μM rGTP, 50 μCi [α-32 P]GTP, and 0.5 mM Ribo m7 G cap analog (Promega).

Concentration Assay:

Article Title: Chemokine Gene Expression in Astrocytes of Borna Disease Virus-Infected Rats and Mice in the Absence of Inflammation
Article Snippet: Purified linearized DNAs were pooled at a concentration of 50 ng each per μl. .. The radiolabeled antisense RPA probe set was synthesized in a volume of 20 μl containing 100 μCi of [α-32 P]UTP (3,000 Ci/mmol); dithiothreitol (200 nmol); transcription buffer (Promega); 1 μl of template DNA; rUTP (61 pmol); rGTP, rATP, and rCTP (2.75 nmol each); RNase inhibitor (28 U; Pharmacia); and T7 polymerase (20 U; Promega).

Lysis:

Article Title: Stem-Loop IV in the 5? Untranslated Region Is a cis-Acting Element in Bovine Coronavirus Defective Interfering RNA Replication
Article Snippet: The lysate was clarified at 700× g at 4°C for 10 min, the protein content, measured with a Bradford kit (Bio-Rad), was adjusted to 20 μg/10 μl with lysis buffer, and 10-μl aliquots were stored at −80°C. .. For synthesis of uniformly radiolabeled RNA probe, plasmid DNA of wild-type (wt) pSLIV(PB) or the PB modifications of stem-loop IV mutants (Table ) was linearized with NcoI, and 2.5 μg was transcribed in a 50-μl reaction volume with 40 U T7 RNA polymerase (Promega) at 37°C for 1 h in the presence of 120 μCi [α-32 P]UTP (300 Ci/mmole; ICN), 0.5 mM each of rATP, rCTP, and rGTP (Promega), 12 μM UTP (Promega), 10 mM DTT, and 5 mg acetylated bovine serum albumin (Promega).

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