Structured Review

Promega rgtp
Impact of MnCl 2 , pH and temperature on calicivirus <t>RdRp</t> activity. Purified recombinant RdRps (40 ng of RHDV and RCV RdRps and 200 ng of NoV RdRp) were used to generate dsRNA with poly(C) RNA (20 ng/μL) as template and <t>rGTP</t> (0.5 mM) as substrate. Following a 15-min incubation in the presence of different ( a ) MnCl 2 concentrations; ( b ) pH levels; or ( c ) temperatures, reactions were stopped with EDTA at a final concentration 5 mM and dsRNA was quantified using the PicoGreen reagent. Unless indicated otherwise, reactions were incubated at 30 °C. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares and NoV RdRp activity is shown in black as circles. Averages of relative fluorescence levels were calculated and plotted with standard deviations. The results were generated from two ( a ) or three ( b and c ) independent experiments with triplicate reactions for each measurement point.
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Images

1) Product Images from "Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors"

Article Title: Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors

Journal: Viruses

doi: 10.3390/v8040100

Impact of MnCl 2 , pH and temperature on calicivirus RdRp activity. Purified recombinant RdRps (40 ng of RHDV and RCV RdRps and 200 ng of NoV RdRp) were used to generate dsRNA with poly(C) RNA (20 ng/μL) as template and rGTP (0.5 mM) as substrate. Following a 15-min incubation in the presence of different ( a ) MnCl 2 concentrations; ( b ) pH levels; or ( c ) temperatures, reactions were stopped with EDTA at a final concentration 5 mM and dsRNA was quantified using the PicoGreen reagent. Unless indicated otherwise, reactions were incubated at 30 °C. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares and NoV RdRp activity is shown in black as circles. Averages of relative fluorescence levels were calculated and plotted with standard deviations. The results were generated from two ( a ) or three ( b and c ) independent experiments with triplicate reactions for each measurement point.
Figure Legend Snippet: Impact of MnCl 2 , pH and temperature on calicivirus RdRp activity. Purified recombinant RdRps (40 ng of RHDV and RCV RdRps and 200 ng of NoV RdRp) were used to generate dsRNA with poly(C) RNA (20 ng/μL) as template and rGTP (0.5 mM) as substrate. Following a 15-min incubation in the presence of different ( a ) MnCl 2 concentrations; ( b ) pH levels; or ( c ) temperatures, reactions were stopped with EDTA at a final concentration 5 mM and dsRNA was quantified using the PicoGreen reagent. Unless indicated otherwise, reactions were incubated at 30 °C. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares and NoV RdRp activity is shown in black as circles. Averages of relative fluorescence levels were calculated and plotted with standard deviations. The results were generated from two ( a ) or three ( b and c ) independent experiments with triplicate reactions for each measurement point.

Techniques Used: Activity Assay, Purification, Recombinant, Incubation, Concentration Assay, Fluorescence, Generated

Comparison of RHDV and RCV RdRp activities. Purified recombinant RdRps were used to generate dsRNA from a poly(C) RNA (20 ng/μL) template using rGTP (0.5 mM) as substrate. Following incubation at 30 °C, reactions were stopped with 5 mM EDTA and dsRNA was quantified using the fluorescent dye PicoGreen. RHDV RdRp activity is shown in red, RCV RdRp activity is shown in blue. The results from a representative experiment are shown with average values and standard deviations from triplicate reactions for each measurement point. ( a ) The effect of RHDV and RCV RdRp concentrations on dsRNA formation over 15 min; ( b ) The synthesis of dsRNA catalysed by 20 ng of RHDV and RCV RdRps over a 1-h period. RFU—relative fluorescence units.
Figure Legend Snippet: Comparison of RHDV and RCV RdRp activities. Purified recombinant RdRps were used to generate dsRNA from a poly(C) RNA (20 ng/μL) template using rGTP (0.5 mM) as substrate. Following incubation at 30 °C, reactions were stopped with 5 mM EDTA and dsRNA was quantified using the fluorescent dye PicoGreen. RHDV RdRp activity is shown in red, RCV RdRp activity is shown in blue. The results from a representative experiment are shown with average values and standard deviations from triplicate reactions for each measurement point. ( a ) The effect of RHDV and RCV RdRp concentrations on dsRNA formation over 15 min; ( b ) The synthesis of dsRNA catalysed by 20 ng of RHDV and RCV RdRps over a 1-h period. RFU—relative fluorescence units.

Techniques Used: Purification, Recombinant, Incubation, Activity Assay, Fluorescence

Kinetics of template and substrate utilisation by calicivirus RdRps. Purified recombinant RdRps (40 ng of RHDV and RCV RdRps and 200 ng of NoV RdRp) were used to generate dsRNA with poly(C) RNA as template and rGTP as substrate. ( a ) The kinetics of template utilisation was examined by titrating poly(C) from 0 to 40 ng/μL in the presence of 0.5 mM rGTP; ( b , c ) The kinetics of nucleotide incorporation was examined by titrating rGTP from 0 to 0.5 mM and 0 to 2.0 mM in the presence of 20 ng/μL of poly(C). Following a 15-min incubation at 30 °C, reactions were stopped with EDTA at final concentration 5 mM and dsRNA was quantified using the PicoGreen reagent. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares and NoV RdRp activity is shown in black as circles. The results from a representative experiment are shown with average values and standard deviations from triplicate reactions for each measurement point.
Figure Legend Snippet: Kinetics of template and substrate utilisation by calicivirus RdRps. Purified recombinant RdRps (40 ng of RHDV and RCV RdRps and 200 ng of NoV RdRp) were used to generate dsRNA with poly(C) RNA as template and rGTP as substrate. ( a ) The kinetics of template utilisation was examined by titrating poly(C) from 0 to 40 ng/μL in the presence of 0.5 mM rGTP; ( b , c ) The kinetics of nucleotide incorporation was examined by titrating rGTP from 0 to 0.5 mM and 0 to 2.0 mM in the presence of 20 ng/μL of poly(C). Following a 15-min incubation at 30 °C, reactions were stopped with EDTA at final concentration 5 mM and dsRNA was quantified using the PicoGreen reagent. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares and NoV RdRp activity is shown in black as circles. The results from a representative experiment are shown with average values and standard deviations from triplicate reactions for each measurement point.

Techniques Used: Purification, Recombinant, Incubation, Concentration Assay, Activity Assay

2) Product Images from "Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors"

Article Title: Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors

Journal: Viruses

doi: 10.3390/v8040100

Impact of MnCl 2 , pH and temperature on calicivirus RdRp activity. Purified recombinant RdRps (40 ng of RHDV and RCV RdRps and 200 ng of NoV RdRp) were used to generate dsRNA with poly(C) RNA (20 ng/μL) as template and rGTP (0.5 mM) as substrate. Following a 15-min incubation in the presence of different ( a ) MnCl 2 concentrations; ( b ) pH levels; or ( c ) temperatures, reactions were stopped with EDTA at a final concentration 5 mM and dsRNA was quantified using the PicoGreen reagent. Unless indicated otherwise, reactions were incubated at 30 °C. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares and NoV RdRp activity is shown in black as circles. Averages of relative fluorescence levels were calculated and plotted with standard deviations. The results were generated from two ( a ) or three ( b and c ) independent experiments with triplicate reactions for each measurement point.
Figure Legend Snippet: Impact of MnCl 2 , pH and temperature on calicivirus RdRp activity. Purified recombinant RdRps (40 ng of RHDV and RCV RdRps and 200 ng of NoV RdRp) were used to generate dsRNA with poly(C) RNA (20 ng/μL) as template and rGTP (0.5 mM) as substrate. Following a 15-min incubation in the presence of different ( a ) MnCl 2 concentrations; ( b ) pH levels; or ( c ) temperatures, reactions were stopped with EDTA at a final concentration 5 mM and dsRNA was quantified using the PicoGreen reagent. Unless indicated otherwise, reactions were incubated at 30 °C. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares and NoV RdRp activity is shown in black as circles. Averages of relative fluorescence levels were calculated and plotted with standard deviations. The results were generated from two ( a ) or three ( b and c ) independent experiments with triplicate reactions for each measurement point.

Techniques Used: Activity Assay, Purification, Recombinant, Incubation, Concentration Assay, Fluorescence, Generated

Comparison of RHDV and RCV RdRp activities. Purified recombinant RdRps were used to generate dsRNA from a poly(C) RNA (20 ng/μL) template using rGTP (0.5 mM) as substrate. Following incubation at 30 °C, reactions were stopped with 5 mM EDTA and dsRNA was quantified using the fluorescent dye PicoGreen. RHDV RdRp activity is shown in red, RCV RdRp activity is shown in blue. The results from a representative experiment are shown with average values and standard deviations from triplicate reactions for each measurement point. ( a ) The effect of RHDV and RCV RdRp concentrations on dsRNA formation over 15 min; ( b ) The synthesis of dsRNA catalysed by 20 ng of RHDV and RCV RdRps over a 1-h period. RFU—relative fluorescence units.
Figure Legend Snippet: Comparison of RHDV and RCV RdRp activities. Purified recombinant RdRps were used to generate dsRNA from a poly(C) RNA (20 ng/μL) template using rGTP (0.5 mM) as substrate. Following incubation at 30 °C, reactions were stopped with 5 mM EDTA and dsRNA was quantified using the fluorescent dye PicoGreen. RHDV RdRp activity is shown in red, RCV RdRp activity is shown in blue. The results from a representative experiment are shown with average values and standard deviations from triplicate reactions for each measurement point. ( a ) The effect of RHDV and RCV RdRp concentrations on dsRNA formation over 15 min; ( b ) The synthesis of dsRNA catalysed by 20 ng of RHDV and RCV RdRps over a 1-h period. RFU—relative fluorescence units.

Techniques Used: Purification, Recombinant, Incubation, Activity Assay, Fluorescence

Kinetics of template and substrate utilisation by calicivirus RdRps. Purified recombinant RdRps (40 ng of RHDV and RCV RdRps and 200 ng of NoV RdRp) were used to generate dsRNA with poly(C) RNA as template and rGTP as substrate. ( a ) The kinetics of template utilisation was examined by titrating poly(C) from 0 to 40 ng/μL in the presence of 0.5 mM rGTP; ( b , c ) The kinetics of nucleotide incorporation was examined by titrating rGTP from 0 to 0.5 mM and 0 to 2.0 mM in the presence of 20 ng/μL of poly(C). Following a 15-min incubation at 30 °C, reactions were stopped with EDTA at final concentration 5 mM and dsRNA was quantified using the PicoGreen reagent. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares and NoV RdRp activity is shown in black as circles. The results from a representative experiment are shown with average values and standard deviations from triplicate reactions for each measurement point.
Figure Legend Snippet: Kinetics of template and substrate utilisation by calicivirus RdRps. Purified recombinant RdRps (40 ng of RHDV and RCV RdRps and 200 ng of NoV RdRp) were used to generate dsRNA with poly(C) RNA as template and rGTP as substrate. ( a ) The kinetics of template utilisation was examined by titrating poly(C) from 0 to 40 ng/μL in the presence of 0.5 mM rGTP; ( b , c ) The kinetics of nucleotide incorporation was examined by titrating rGTP from 0 to 0.5 mM and 0 to 2.0 mM in the presence of 20 ng/μL of poly(C). Following a 15-min incubation at 30 °C, reactions were stopped with EDTA at final concentration 5 mM and dsRNA was quantified using the PicoGreen reagent. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares and NoV RdRp activity is shown in black as circles. The results from a representative experiment are shown with average values and standard deviations from triplicate reactions for each measurement point.

Techniques Used: Purification, Recombinant, Incubation, Concentration Assay, Activity Assay

Related Articles

Clone Assay:

Article Title: Characterization of recombinant Saccharomyces cerevisiae telomerase core enzyme purified from yeast
Article Snippet: T7 RNA polymerase promoter sequence was cloned into the pUC19 vector by annealing the two oligonucleotides 5′-CGGGGTACCGTAATACGACTCACTATAGGGATCCCG-3′ and 5′-CGGGATCCCTATAGTGAGTCGTATTACGGTACCCCG-3′ and digesting with KpnI and BamHI, resulting in the pUC19-T7 plasmid. .. Tlc1 in vitro transcription was carried out in a 20 μl reaction volume containing 1 μg of DNA template, 2.5 mM each of rATP, rCTP, rTTP and rGTP (Promega), 0.05 unit of pyrophosphatase (Sigma), 40 units of recombinant RNasin, 8 mM GMP (Sigma) and 700 units of T7 RNA polymerase (kindly provided by Professor En-Duo Wang's laboratory in our institute) in transcription buffer (40 mM Tris/HCl, pH 8.0, 5 mM DTT, 30 mM MgCl2 , 1 mM spermidine and 0.1% Triton X-100) for 4 h at 30 °C.

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: RNA Directed Modulation of Phenotypic Plasticity in Human Cells
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Amplification:

Article Title: Long-Term Follow-Up of Chimpanzees Inoculated with the First Infectious Clone for Hepatitis C Virus
Article Snippet: Duplicate RNA samples were amplified with the TaqMan EZ RT-PCR kit and a TaqMan fluorogenic probe labeled with FAM (6-carboxy-fluorescein) and TAMRA (6-carboxytetramethylrhodamine) (PE-Applied Biosystems) at the 5′ and 3′ ends, respectively. .. The 100-μl reaction mixture, containing 20 μl of 5× T7 buffer; 7.5 mM (each) rATP, rCTP, rGTP, and rUTP; 2 μg of DNA; and 10 μl of enzyme mixture was incubated at 37°C for 2 h and then treated with 10 U of RNase-free DNase (Promega) for 30 min at 37°C.

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: RNA Directed Modulation of Phenotypic Plasticity in Human Cells
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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: 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.

Real-time Polymerase Chain Reaction:

Article Title: Characterization of Multiple Exon 1 Variants in Mammalian HuD mRNA and Neuron-Specific Transcriptional Control via Neurogenin 2
Article Snippet: Nuclei were split into two aliquots and incubated in a transcription reaction mix containing 20% glycerol, 30 m m Tris-HCl pH 8.0, 2.5 m m MgCl2 , 150 m m KCl, 1 m m DTT, 40U of Rnasin and 0.5 m m rATP, rCTP, rGTP, and rUTP (+) or diethylpyrocarbonate (DEPC)-treated water (−) (Promega). .. Reactions were incubated for 30 min at 30°C, then RNA was TRIzol extracted and real-time qPCR was performed with HuD and 18S primers.

Quantitation Assay:

Article Title: Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors
Article Snippet: A standard reaction contained 20 ng/μL poly(C) RNA (Sigma-Aldrich), 0.5 mM rGTP (Promega), 2.5 mM MnCl2 , and 5 mM DTT in 20 mM Tris-HCl (pH 7.5) buffer. .. Reactions were terminated with 5 mM EDTA, followed by PicoGreen staining and dsRNA quantitation using either POLARstar or SpectraMax M3 plate readers at standard wavelengths (excitation 480 nm, emission 520 nm).

Stripping Membranes:

Article Title: RNA Directed Modulation of Phenotypic Plasticity in Human Cells
Article Snippet: .. Generating Biotin Labeled RNA Transcripts- Biotin labeled RNA transcripts of Retro-EFI2S2 and GFP were generated by adding the following components into an 8 well PCR strip: (2.5μg of DNA template, 2μl T7 Enzyme mix (Sigma), 1ul rATP, rGTP, rCTP, rUTP (Promega), 1μl Biotin-14-CTP (Life Technologies), 2.5μl 1M DTT (Sigma) and 1μl RNase Inhibitor (Life Technologies)(as described in [ ]). .. The reaction volume was made up to 50μl with RNase free water (QIAGEN).

Activity Assay:

Article Title: Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors
Article Snippet: Paragraph title: 2.4. Kinetics of RdRp Activity ... A standard reaction contained 20 ng/μL poly(C) RNA (Sigma-Aldrich), 0.5 mM rGTP (Promega), 2.5 mM MnCl2 , and 5 mM DTT in 20 mM Tris-HCl (pH 7.5) buffer.

Article Title: Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors
Article Snippet: The new template PE46-C (5′-CCCCCCCCCCCCCCCAUAUACUUCGGUAUAUGG-3′) was used to direct primer extension activity through a stable hairpin at the 3′ end. .. Reactions were performed using 1 µM PE46-C template and 400 ng RdRp, 0.4 mM rGTP (Promega), 5 mM DTT, 2.5 mM MnCl2 , and 20 mM Tris-HCl (pH 7.5) in a final volume of 25 µL.

Expressing:

Article Title: RNA Directed Modulation of Phenotypic Plasticity in Human Cells
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Article Title: Characterization of Multiple Exon 1 Variants in Mammalian HuD mRNA and Neuron-Specific Transcriptional Control via Neurogenin 2
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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).

Modification:

Article Title: Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors
Article Snippet: The original template PE46 was modified to replace 13 nucleotides at the 5′ end with 13 cytosines, resulting in a new template PE46-C. .. Reactions were performed using 1 µM PE46-C template and 400 ng RdRp, 0.4 mM rGTP (Promega), 5 mM DTT, 2.5 mM MnCl2 , and 20 mM Tris-HCl (pH 7.5) in a final volume of 25 µL.

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.

Hybridization:

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].

Transfection:

Article Title: RNA Directed Modulation of Phenotypic Plasticity in Human Cells
Article Snippet: Midi prepped (QIAGEN plasmid Midi Kit) clones were transfected along with the pcDNA3.1-GFP control [ ] into target cells using lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). .. Generating Biotin Labeled RNA Transcripts- Biotin labeled RNA transcripts of Retro-EFI2S2 and GFP were generated by adding the following components into an 8 well PCR strip: (2.5μg of DNA template, 2μl T7 Enzyme mix (Sigma), 1ul rATP, rGTP, rCTP, rUTP (Promega), 1μl Biotin-14-CTP (Life Technologies), 2.5μl 1M DTT (Sigma) and 1μl RNase Inhibitor (Life Technologies)(as described in [ ]).

Serial Dilution:

Article Title: Long-Term Follow-Up of Chimpanzees Inoculated with the First Infectious Clone for Hepatitis C Virus
Article Snippet: The 100-μl reaction mixture, containing 20 μl of 5× T7 buffer; 7.5 mM (each) rATP, rCTP, rGTP, and rUTP; 2 μg of DNA; and 10 μl of enzyme mixture was incubated at 37°C for 2 h and then treated with 10 U of RNase-free DNase (Promega) for 30 min at 37°C. .. Standards of 105 , 104 , 103 , 102 , 101 , and 100 RNA copies/μl were prepared by serial dilution of the stock RNA in RNasin-DTT water containing 10 μg of tRNA/ml and stored at −80°C.

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: RNA Directed Modulation of Phenotypic Plasticity in Human Cells
Article Snippet: .. Generating Biotin Labeled RNA Transcripts- Biotin labeled RNA transcripts of Retro-EFI2S2 and GFP were generated by adding the following components into an 8 well PCR strip: (2.5μg of DNA template, 2μl T7 Enzyme mix (Sigma), 1ul rATP, rGTP, rCTP, rUTP (Promega), 1μl Biotin-14-CTP (Life Technologies), 2.5μl 1M DTT (Sigma) and 1μl RNase Inhibitor (Life Technologies)(as described in [ ]). .. The reaction volume was made up to 50μl with RNase free water (QIAGEN).

Polymerase Chain Reaction:

Article Title: RNA Directed Modulation of Phenotypic Plasticity in Human Cells
Article Snippet: .. Generating Biotin Labeled RNA Transcripts- Biotin labeled RNA transcripts of Retro-EFI2S2 and GFP were generated by adding the following components into an 8 well PCR strip: (2.5μg of DNA template, 2μl T7 Enzyme mix (Sigma), 1ul rATP, rGTP, rCTP, rUTP (Promega), 1μl Biotin-14-CTP (Life Technologies), 2.5μl 1M DTT (Sigma) and 1μl RNase Inhibitor (Life Technologies)(as described in [ ]). .. The reaction volume was made up to 50μl with RNase free water (QIAGEN).

Sequencing:

Article Title: Characterization of recombinant Saccharomyces cerevisiae telomerase core enzyme purified from yeast
Article Snippet: T7 RNA polymerase promoter sequence was cloned into the pUC19 vector by annealing the two oligonucleotides 5′-CGGGGTACCGTAATACGACTCACTATAGGGATCCCG-3′ and 5′-CGGGATCCCTATAGTGAGTCGTATTACGGTACCCCG-3′ and digesting with KpnI and BamHI, resulting in the pUC19-T7 plasmid. .. Tlc1 in vitro transcription was carried out in a 20 μl reaction volume containing 1 μg of DNA template, 2.5 mM each of rATP, rCTP, rTTP and rGTP (Promega), 0.05 unit of pyrophosphatase (Sigma), 40 units of recombinant RNasin, 8 mM GMP (Sigma) and 700 units of T7 RNA polymerase (kindly provided by Professor En-Duo Wang's laboratory in our institute) in transcription buffer (40 mM Tris/HCl, pH 8.0, 5 mM DTT, 30 mM MgCl2 , 1 mM spermidine and 0.1% Triton X-100) for 4 h at 30 °C.

Recombinant:

Article Title: Characterization of recombinant Saccharomyces cerevisiae telomerase core enzyme purified from yeast
Article Snippet: .. Tlc1 in vitro transcription was carried out in a 20 μl reaction volume containing 1 μg of DNA template, 2.5 mM each of rATP, rCTP, rTTP and rGTP (Promega), 0.05 unit of pyrophosphatase (Sigma), 40 units of recombinant RNasin, 8 mM GMP (Sigma) and 700 units of T7 RNA polymerase (kindly provided by Professor En-Duo Wang's laboratory in our institute) in transcription buffer (40 mM Tris/HCl, pH 8.0, 5 mM DTT, 30 mM MgCl2 , 1 mM spermidine and 0.1% Triton X-100) for 4 h at 30 °C. .. RNase-free DNase (1 unit) (Promega) was used to remove the DNA template at 37 °C for 30 min, followed by heat-inactivation at 70 °C for 10 min.

Article Title: The Andes Hantavirus NSs Protein Is Expressed from the Viral Small mRNA by a Leaky Scanning Mechanism
Article Snippet: .. Linear DNA (1 μg) was used to program in vitro transcription reactions using 3 mM DTT, 0.5 mM MgCl2 , 10 mM rATP, 10 mM recombinant CTP (rCTP), 10 mM rUTP, 2 mM rGTP, Ribomax buffer (Promega), 20 U of RNasin (Promega), 1.28 mM 7 mGpppG cap analogue (Promega), and 10 U of T7 RNA polymerase (Fermentas) at 37°C for 2 h. Template DNA was digested with DNase RQ1 (Promega), and the RNA was precipitated with 2.8 M LiCl. .. RNA was resuspended in nuclease-free water and its concentration determined spectrophotometrically by a Nanodrop (Nanodrop Technology, Wilmington, DE).

Article Title: Long-Term Follow-Up of Chimpanzees Inoculated with the First Infectious Clone for Hepatitis C Virus
Article Snippet: Reactions were performed with 50-μl mixtures containing the following: 5 μl of template RNA; 10 μl of 5× TaqMan EZ buffer; 2.5 mM Mn(OAc)2 ; 300 μM (each) dATP, dCTP, dGTP, and dTTP; 300 nM (each) primers TaqMan/243 and TaqMan/390; 100 nM fluorogenic probe (TaqMan/336); and 5 U of recombinant Tth (r Tth ) DNA polymerase. .. The 100-μl reaction mixture, containing 20 μl of 5× T7 buffer; 7.5 mM (each) rATP, rCTP, rGTP, and rUTP; 2 μg of DNA; and 10 μl of enzyme mixture was incubated at 37°C for 2 h and then treated with 10 U of RNase-free DNase (Promega) for 30 min at 37°C.

Fluorescence:

Article Title: Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors
Article Snippet: A standard reaction contained 20 ng/μL poly(C) RNA (Sigma-Aldrich), 0.5 mM rGTP (Promega), 2.5 mM MnCl2 , and 5 mM DTT in 20 mM Tris-HCl (pH 7.5) buffer. .. Control reactions stopped at 0 min were used to quantify background fluorescence.

Labeling:

Article Title: RNA Directed Modulation of Phenotypic Plasticity in Human Cells
Article Snippet: .. Generating Biotin Labeled RNA Transcripts- Biotin labeled RNA transcripts of Retro-EFI2S2 and GFP were generated by adding the following components into an 8 well PCR strip: (2.5μg of DNA template, 2μl T7 Enzyme mix (Sigma), 1ul rATP, rGTP, rCTP, rUTP (Promega), 1μl Biotin-14-CTP (Life Technologies), 2.5μl 1M DTT (Sigma) and 1μl RNase Inhibitor (Life Technologies)(as described in [ ]). .. The reaction volume was made up to 50μl with RNase free water (QIAGEN).

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: Long-Term Follow-Up of Chimpanzees Inoculated with the First Infectious Clone for Hepatitis C Virus
Article Snippet: Duplicate RNA samples were amplified with the TaqMan EZ RT-PCR kit and a TaqMan fluorogenic probe labeled with FAM (6-carboxy-fluorescein) and TAMRA (6-carboxytetramethylrhodamine) (PE-Applied Biosystems) at the 5′ and 3′ ends, respectively. .. The 100-μl reaction mixture, containing 20 μl of 5× T7 buffer; 7.5 mM (each) rATP, rCTP, rGTP, and rUTP; 2 μg of DNA; and 10 μl of enzyme mixture was incubated at 37°C for 2 h and then treated with 10 U of RNase-free DNase (Promega) for 30 min at 37°C.

Article Title: Array-Based Nuclear Run-on (ANRO) Analysis
Article Snippet: Paragraph title: 2.3. Materials and Reagents for Nascent RNA Labeling and RNA Purification ... 2× NRO Reaction Buffer: 300 mM KCl, 10 mM MgCl2 , 2 mM each of rATP, rCTP, rGTP (Promega cat. # E6000), 1 mM Bio-16-UTP (Enzo Life Sciences, cat. # ENZ-42814), 800 Units/ml RNaseOUT (Invitrogen cat. # 10777-019). rUTP (Promega cat. # 6000).

Purification:

Article Title: Characterization of recombinant Saccharomyces cerevisiae telomerase core enzyme purified from yeast
Article Snippet: Tlc1 in vitro transcription was carried out in a 20 μl reaction volume containing 1 μg of DNA template, 2.5 mM each of rATP, rCTP, rTTP and rGTP (Promega), 0.05 unit of pyrophosphatase (Sigma), 40 units of recombinant RNasin, 8 mM GMP (Sigma) and 700 units of T7 RNA polymerase (kindly provided by Professor En-Duo Wang's laboratory in our institute) in transcription buffer (40 mM Tris/HCl, pH 8.0, 5 mM DTT, 30 mM MgCl2 , 1 mM spermidine and 0.1% Triton X-100) for 4 h at 30 °C. .. The transcripts were then purified with RNeasy Minielute Cleanup Kit (Qiagen).

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).

Article Title: Array-Based Nuclear Run-on (ANRO) Analysis
Article Snippet: Paragraph title: 2.3. Materials and Reagents for Nascent RNA Labeling and RNA Purification ... 2× NRO Reaction Buffer: 300 mM KCl, 10 mM MgCl2 , 2 mM each of rATP, rCTP, rGTP (Promega cat. # E6000), 1 mM Bio-16-UTP (Enzo Life Sciences, cat. # ENZ-42814), 800 Units/ml RNaseOUT (Invitrogen cat. # 10777-019). rUTP (Promega cat. # 6000).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Long-Term Follow-Up of Chimpanzees Inoculated with the First Infectious Clone for Hepatitis C Virus
Article Snippet: Duplicate RNA samples were amplified with the TaqMan EZ RT-PCR kit and a TaqMan fluorogenic probe labeled with FAM (6-carboxy-fluorescein) and TAMRA (6-carboxytetramethylrhodamine) (PE-Applied Biosystems) at the 5′ and 3′ ends, respectively. .. The 100-μl reaction mixture, containing 20 μl of 5× T7 buffer; 7.5 mM (each) rATP, rCTP, rGTP, and rUTP; 2 μg of DNA; and 10 μl of enzyme mixture was incubated at 37°C for 2 h and then treated with 10 U of RNase-free DNase (Promega) for 30 min at 37°C.

Protein Extraction:

Article Title: Characterization of Multiple Exon 1 Variants in Mammalian HuD mRNA and Neuron-Specific Transcriptional Control via Neurogenin 2
Article Snippet: Briefly, nuclear extracts were harvested from neuron or mesoderm differentiated P19 cells using the ProteoJet Nuclear and Cytoplasmic Protein Extraction kit (Fermentas), according to manufacturer's protocol. .. Nuclei were split into two aliquots and incubated in a transcription reaction mix containing 20% glycerol, 30 m m Tris-HCl pH 8.0, 2.5 m m MgCl2 , 150 m m KCl, 1 m m DTT, 40U of Rnasin and 0.5 m m rATP, rCTP, rGTP, and rUTP (+) or diethylpyrocarbonate (DEPC)-treated water (−) (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).

Staining:

Article Title: Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors
Article Snippet: A standard reaction contained 20 ng/μL poly(C) RNA (Sigma-Aldrich), 0.5 mM rGTP (Promega), 2.5 mM MnCl2 , and 5 mM DTT in 20 mM Tris-HCl (pH 7.5) buffer. .. Reactions were terminated with 5 mM EDTA, followed by PicoGreen staining and dsRNA quantitation using either POLARstar or SpectraMax M3 plate readers at standard wavelengths (excitation 480 nm, emission 520 nm).

Plasmid Preparation:

Article Title: Characterization of recombinant Saccharomyces cerevisiae telomerase core enzyme purified from yeast
Article Snippet: The DNA template for in vitro transcription was created by linearizing the pUC19-T7- TLC1 plasmid using XbaI, and the ends were blunted using T4 DNA polymerase. .. Tlc1 in vitro transcription was carried out in a 20 μl reaction volume containing 1 μg of DNA template, 2.5 mM each of rATP, rCTP, rTTP and rGTP (Promega), 0.05 unit of pyrophosphatase (Sigma), 40 units of recombinant RNasin, 8 mM GMP (Sigma) and 700 units of T7 RNA polymerase (kindly provided by Professor En-Duo Wang's laboratory in our institute) in transcription buffer (40 mM Tris/HCl, pH 8.0, 5 mM DTT, 30 mM MgCl2 , 1 mM spermidine and 0.1% Triton X-100) for 4 h at 30 °C.

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: RNA Directed Modulation of Phenotypic Plasticity in Human Cells
Article Snippet: Midi prepped (QIAGEN plasmid Midi Kit) clones were transfected along with the pcDNA3.1-GFP control [ ] into target cells using lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). .. Generating Biotin Labeled RNA Transcripts- Biotin labeled RNA transcripts of Retro-EFI2S2 and GFP were generated by adding the following components into an 8 well PCR strip: (2.5μg of DNA template, 2μl T7 Enzyme mix (Sigma), 1ul rATP, rGTP, rCTP, rUTP (Promega), 1μl Biotin-14-CTP (Life Technologies), 2.5μl 1M DTT (Sigma) and 1μl RNase Inhibitor (Life Technologies)(as described in [ ]).

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]).

Electrophoresis:

Article Title: The Andes Hantavirus NSs Protein Is Expressed from the Viral Small mRNA by a Leaky Scanning Mechanism
Article Snippet: Linear DNA (1 μg) was used to program in vitro transcription reactions using 3 mM DTT, 0.5 mM MgCl2 , 10 mM rATP, 10 mM recombinant CTP (rCTP), 10 mM rUTP, 2 mM rGTP, Ribomax buffer (Promega), 20 U of RNasin (Promega), 1.28 mM 7 mGpppG cap analogue (Promega), and 10 U of T7 RNA polymerase (Fermentas) at 37°C for 2 h. Template DNA was digested with DNase RQ1 (Promega), and the RNA was precipitated with 2.8 M LiCl. .. RNA integrity was confirmed by electrophoresis on denaturing agarose gels.

In Vitro:

Article Title: Characterization of recombinant Saccharomyces cerevisiae telomerase core enzyme purified from yeast
Article Snippet: .. Tlc1 in vitro transcription was carried out in a 20 μl reaction volume containing 1 μg of DNA template, 2.5 mM each of rATP, rCTP, rTTP and rGTP (Promega), 0.05 unit of pyrophosphatase (Sigma), 40 units of recombinant RNasin, 8 mM GMP (Sigma) and 700 units of T7 RNA polymerase (kindly provided by Professor En-Duo Wang's laboratory in our institute) in transcription buffer (40 mM Tris/HCl, pH 8.0, 5 mM DTT, 30 mM MgCl2 , 1 mM spermidine and 0.1% Triton X-100) for 4 h at 30 °C. .. RNase-free DNase (1 unit) (Promega) was used to remove the DNA template at 37 °C for 30 min, followed by heat-inactivation at 70 °C for 10 min.

Article Title: The extruded non-template strand determines the architecture of R-loops
Article Snippet: .. In vitro transcription of R-loops 1875 ng of circular plasmids were incubated at 37°C with 50 U of T3 RNA Polymerase (Promega) in a transcription buffer (40 mM Tris–HCl pH 7.9, 6 mM MgCl2 , 2 mM spermidine, 10 mM NaCl, 20 mM DTT, 0,05% Tween-20) containing 0,25 mM of rATP, rCTP, rUTP and rGTP (Promega). ..

Article Title: Substitutions in Bacteriophage T4 AsiA and Escherichia coli ?70 That Suppress T4 motA Activation Mutations
Article Snippet: Paragraph title: In vitro transcription. ... Transcription assays (10 μl) also contained 400 μM (each) rATP, rGTP, and rCTP, 40 μM UTP, 4 U of RNasin (Promega), 25 to 50 fmol of DNA template, and 0.7 μM [α-32 P]UTP (3,000 Ci/mmol), along with the indicated proteins.

Article Title: The Andes Hantavirus NSs Protein Is Expressed from the Viral Small mRNA by a Leaky Scanning Mechanism
Article Snippet: .. Linear DNA (1 μg) was used to program in vitro transcription reactions using 3 mM DTT, 0.5 mM MgCl2 , 10 mM rATP, 10 mM recombinant CTP (rCTP), 10 mM rUTP, 2 mM rGTP, Ribomax buffer (Promega), 20 U of RNasin (Promega), 1.28 mM 7 mGpppG cap analogue (Promega), and 10 U of T7 RNA polymerase (Fermentas) at 37°C for 2 h. Template DNA was digested with DNase RQ1 (Promega), and the RNA was precipitated with 2.8 M LiCl. .. RNA was resuspended in nuclease-free water and its concentration determined spectrophotometrically by a Nanodrop (Nanodrop Technology, Wilmington, DE).

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).

Incubation:

Article Title: Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors
Article Snippet: A standard reaction contained 20 ng/μL poly(C) RNA (Sigma-Aldrich), 0.5 mM rGTP (Promega), 2.5 mM MnCl2 , and 5 mM DTT in 20 mM Tris-HCl (pH 7.5) buffer. .. Reactions were initiated by the addition of 20–40 ng RdRp and incubated at 30 °C for 15 min unless stated otherwise.

Article Title: Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors
Article Snippet: Reactions were performed using 1 µM PE46-C template and 400 ng RdRp, 0.4 mM rGTP (Promega), 5 mM DTT, 2.5 mM MnCl2 , and 20 mM Tris-HCl (pH 7.5) in a final volume of 25 µL. .. RdRps were incubated for 10 min at 30 °C in the presence of the test compounds or the compound vehicle DMSO (0.5% v/v ) prior to addition into the reaction mixture.

Article Title: The extruded non-template strand determines the architecture of R-loops
Article Snippet: .. In vitro transcription of R-loops 1875 ng of circular plasmids were incubated at 37°C with 50 U of T3 RNA Polymerase (Promega) in a transcription buffer (40 mM Tris–HCl pH 7.9, 6 mM MgCl2 , 2 mM spermidine, 10 mM NaCl, 20 mM DTT, 0,05% Tween-20) containing 0,25 mM of rATP, rCTP, rUTP and rGTP (Promega). ..

Article Title: Characterization of Multiple Exon 1 Variants in Mammalian HuD mRNA and Neuron-Specific Transcriptional Control via Neurogenin 2
Article Snippet: .. Nuclei were split into two aliquots and incubated in a transcription reaction mix containing 20% glycerol, 30 m m Tris-HCl pH 8.0, 2.5 m m MgCl2 , 150 m m KCl, 1 m m DTT, 40U of Rnasin and 0.5 m m rATP, rCTP, rGTP, and rUTP (+) or diethylpyrocarbonate (DEPC)-treated water (−) (Promega). .. Reactions were incubated for 30 min at 30°C, then RNA was TRIzol extracted and real-time qPCR was performed with HuD and 18S primers.

Article Title: Long-Term Follow-Up of Chimpanzees Inoculated with the First Infectious Clone for Hepatitis C Virus
Article Snippet: .. The 100-μl reaction mixture, containing 20 μl of 5× T7 buffer; 7.5 mM (each) rATP, rCTP, rGTP, and rUTP; 2 μg of DNA; and 10 μl of enzyme mixture was incubated at 37°C for 2 h and then treated with 10 U of RNase-free DNase (Promega) for 30 min at 37°C. ..

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).

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).

Article Title: Long-Term Follow-Up of Chimpanzees Inoculated with the First Infectious Clone for Hepatitis C Virus
Article Snippet: RNA standards, run in duplicate in every reaction, were prepared from RNA transcripts produced with the RiboMAX T7 large-scale RNA production system (Promega) and the p90 full-length plasmid linearized with Bsm I. .. The 100-μl reaction mixture, containing 20 μl of 5× T7 buffer; 7.5 mM (each) rATP, rCTP, rGTP, and rUTP; 2 μg of DNA; and 10 μl of enzyme mixture was incubated at 37°C for 2 h and then treated with 10 U of RNase-free DNase (Promega) for 30 min at 37°C.

Concentration Assay:

Article Title: The extruded non-template strand determines the architecture of R-loops
Article Snippet: In vitro transcription of R-loops 1875 ng of circular plasmids were incubated at 37°C with 50 U of T3 RNA Polymerase (Promega) in a transcription buffer (40 mM Tris–HCl pH 7.9, 6 mM MgCl2 , 2 mM spermidine, 10 mM NaCl, 20 mM DTT, 0,05% Tween-20) containing 0,25 mM of rATP, rCTP, rUTP and rGTP (Promega). .. After 140 min at 37°C, the NaCl concentration was brought to 500 mM and 0.4 μg of RNase A was added for 20 min at 37°C to digest soluble RNAs.

Article Title: The Andes Hantavirus NSs Protein Is Expressed from the Viral Small mRNA by a Leaky Scanning Mechanism
Article Snippet: Linear DNA (1 μg) was used to program in vitro transcription reactions using 3 mM DTT, 0.5 mM MgCl2 , 10 mM rATP, 10 mM recombinant CTP (rCTP), 10 mM rUTP, 2 mM rGTP, Ribomax buffer (Promega), 20 U of RNasin (Promega), 1.28 mM 7 mGpppG cap analogue (Promega), and 10 U of T7 RNA polymerase (Fermentas) at 37°C for 2 h. Template DNA was digested with DNase RQ1 (Promega), and the RNA was precipitated with 2.8 M LiCl. .. RNA was resuspended in nuclease-free water and its concentration determined spectrophotometrically by a Nanodrop (Nanodrop Technology, Wilmington, DE).

Article Title: Long-Term Follow-Up of Chimpanzees Inoculated with the First Infectious Clone for Hepatitis C Virus
Article Snippet: The 100-μl reaction mixture, containing 20 μl of 5× T7 buffer; 7.5 mM (each) rATP, rCTP, rGTP, and rUTP; 2 μg of DNA; and 10 μl of enzyme mixture was incubated at 37°C for 2 h and then treated with 10 U of RNase-free DNase (Promega) for 30 min at 37°C. .. The concentration was calculated by optical density readings at 260 nm (OD260 ), and the RNA was stored in aliquots of 100 ng in liquid nitrogen until use.

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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    Promega rgtp
    Impact of MnCl 2 , pH and temperature on calicivirus <t>RdRp</t> activity. Purified recombinant RdRps (40 ng of RHDV and RCV RdRps and 200 ng of NoV RdRp) were used to generate dsRNA with poly(C) RNA (20 ng/μL) as template and <t>rGTP</t> (0.5 mM) as substrate. Following a 15-min incubation in the presence of different ( a ) MnCl 2 concentrations; ( b ) pH levels; or ( c ) temperatures, reactions were stopped with EDTA at a final concentration 5 mM and dsRNA was quantified using the PicoGreen reagent. Unless indicated otherwise, reactions were incubated at 30 °C. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares and NoV RdRp activity is shown in black as circles. Averages of relative fluorescence levels were calculated and plotted with standard deviations. The results were generated from two ( a ) or three ( b and c ) independent experiments with triplicate reactions for each measurement point.
    Rgtp, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    rgtp - by Bioz Stars, 2020-01
    94/100 stars
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    Promega a 32 p rgtp
    Impact of MnCl 2 , pH and temperature on calicivirus <t>RdRp</t> activity. Purified recombinant RdRps (40 ng of RHDV and RCV RdRps and 200 ng of NoV RdRp) were used to generate dsRNA with poly(C) RNA (20 ng/μL) as template and <t>rGTP</t> (0.5 mM) as substrate. Following a 15-min incubation in the presence of different ( a ) MnCl 2 concentrations; ( b ) pH levels; or ( c ) temperatures, reactions were stopped with EDTA at a final concentration 5 mM and dsRNA was quantified using the PicoGreen reagent. Unless indicated otherwise, reactions were incubated at 30 °C. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares and NoV RdRp activity is shown in black as circles. Averages of relative fluorescence levels were calculated and plotted with standard deviations. The results were generated from two ( a ) or three ( b and c ) independent experiments with triplicate reactions for each measurement point.
    A 32 P Rgtp, supplied by Promega, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/a 32 p rgtp/product/Promega
    Average 78 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    a 32 p rgtp - by Bioz Stars, 2020-01
    78/100 stars
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    Impact of MnCl 2 , pH and temperature on calicivirus RdRp activity. Purified recombinant RdRps (40 ng of RHDV and RCV RdRps and 200 ng of NoV RdRp) were used to generate dsRNA with poly(C) RNA (20 ng/μL) as template and rGTP (0.5 mM) as substrate. Following a 15-min incubation in the presence of different ( a ) MnCl 2 concentrations; ( b ) pH levels; or ( c ) temperatures, reactions were stopped with EDTA at a final concentration 5 mM and dsRNA was quantified using the PicoGreen reagent. Unless indicated otherwise, reactions were incubated at 30 °C. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares and NoV RdRp activity is shown in black as circles. Averages of relative fluorescence levels were calculated and plotted with standard deviations. The results were generated from two ( a ) or three ( b and c ) independent experiments with triplicate reactions for each measurement point.

    Journal: Viruses

    Article Title: Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors

    doi: 10.3390/v8040100

    Figure Lengend Snippet: Impact of MnCl 2 , pH and temperature on calicivirus RdRp activity. Purified recombinant RdRps (40 ng of RHDV and RCV RdRps and 200 ng of NoV RdRp) were used to generate dsRNA with poly(C) RNA (20 ng/μL) as template and rGTP (0.5 mM) as substrate. Following a 15-min incubation in the presence of different ( a ) MnCl 2 concentrations; ( b ) pH levels; or ( c ) temperatures, reactions were stopped with EDTA at a final concentration 5 mM and dsRNA was quantified using the PicoGreen reagent. Unless indicated otherwise, reactions were incubated at 30 °C. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares and NoV RdRp activity is shown in black as circles. Averages of relative fluorescence levels were calculated and plotted with standard deviations. The results were generated from two ( a ) or three ( b and c ) independent experiments with triplicate reactions for each measurement point.

    Article Snippet: Reactions were performed using 1 µM PE46-C template and 400 ng RdRp, 0.4 mM rGTP (Promega), 5 mM DTT, 2.5 mM MnCl2 , and 20 mM Tris-HCl (pH 7.5) in a final volume of 25 µL.

    Techniques: Activity Assay, Purification, Recombinant, Incubation, Concentration Assay, Fluorescence, Generated

    Comparison of RHDV and RCV RdRp activities. Purified recombinant RdRps were used to generate dsRNA from a poly(C) RNA (20 ng/μL) template using rGTP (0.5 mM) as substrate. Following incubation at 30 °C, reactions were stopped with 5 mM EDTA and dsRNA was quantified using the fluorescent dye PicoGreen. RHDV RdRp activity is shown in red, RCV RdRp activity is shown in blue. The results from a representative experiment are shown with average values and standard deviations from triplicate reactions for each measurement point. ( a ) The effect of RHDV and RCV RdRp concentrations on dsRNA formation over 15 min; ( b ) The synthesis of dsRNA catalysed by 20 ng of RHDV and RCV RdRps over a 1-h period. RFU—relative fluorescence units.

    Journal: Viruses

    Article Title: Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors

    doi: 10.3390/v8040100

    Figure Lengend Snippet: Comparison of RHDV and RCV RdRp activities. Purified recombinant RdRps were used to generate dsRNA from a poly(C) RNA (20 ng/μL) template using rGTP (0.5 mM) as substrate. Following incubation at 30 °C, reactions were stopped with 5 mM EDTA and dsRNA was quantified using the fluorescent dye PicoGreen. RHDV RdRp activity is shown in red, RCV RdRp activity is shown in blue. The results from a representative experiment are shown with average values and standard deviations from triplicate reactions for each measurement point. ( a ) The effect of RHDV and RCV RdRp concentrations on dsRNA formation over 15 min; ( b ) The synthesis of dsRNA catalysed by 20 ng of RHDV and RCV RdRps over a 1-h period. RFU—relative fluorescence units.

    Article Snippet: Reactions were performed using 1 µM PE46-C template and 400 ng RdRp, 0.4 mM rGTP (Promega), 5 mM DTT, 2.5 mM MnCl2 , and 20 mM Tris-HCl (pH 7.5) in a final volume of 25 µL.

    Techniques: Purification, Recombinant, Incubation, Activity Assay, Fluorescence

    Kinetics of template and substrate utilisation by calicivirus RdRps. Purified recombinant RdRps (40 ng of RHDV and RCV RdRps and 200 ng of NoV RdRp) were used to generate dsRNA with poly(C) RNA as template and rGTP as substrate. ( a ) The kinetics of template utilisation was examined by titrating poly(C) from 0 to 40 ng/μL in the presence of 0.5 mM rGTP; ( b , c ) The kinetics of nucleotide incorporation was examined by titrating rGTP from 0 to 0.5 mM and 0 to 2.0 mM in the presence of 20 ng/μL of poly(C). Following a 15-min incubation at 30 °C, reactions were stopped with EDTA at final concentration 5 mM and dsRNA was quantified using the PicoGreen reagent. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares and NoV RdRp activity is shown in black as circles. The results from a representative experiment are shown with average values and standard deviations from triplicate reactions for each measurement point.

    Journal: Viruses

    Article Title: Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors

    doi: 10.3390/v8040100

    Figure Lengend Snippet: Kinetics of template and substrate utilisation by calicivirus RdRps. Purified recombinant RdRps (40 ng of RHDV and RCV RdRps and 200 ng of NoV RdRp) were used to generate dsRNA with poly(C) RNA as template and rGTP as substrate. ( a ) The kinetics of template utilisation was examined by titrating poly(C) from 0 to 40 ng/μL in the presence of 0.5 mM rGTP; ( b , c ) The kinetics of nucleotide incorporation was examined by titrating rGTP from 0 to 0.5 mM and 0 to 2.0 mM in the presence of 20 ng/μL of poly(C). Following a 15-min incubation at 30 °C, reactions were stopped with EDTA at final concentration 5 mM and dsRNA was quantified using the PicoGreen reagent. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares and NoV RdRp activity is shown in black as circles. The results from a representative experiment are shown with average values and standard deviations from triplicate reactions for each measurement point.

    Article Snippet: Reactions were performed using 1 µM PE46-C template and 400 ng RdRp, 0.4 mM rGTP (Promega), 5 mM DTT, 2.5 mM MnCl2 , and 20 mM Tris-HCl (pH 7.5) in a final volume of 25 µL.

    Techniques: Purification, Recombinant, Incubation, Concentration Assay, Activity Assay