All RNAs other than HEV, FCV and radiolabeled transcripts (see RdRp assay protocol) were generated as follows. Five micrograms of linearized plasmid template were pretreated with RNAsecure (Thermo Fisher) and used in a 50 µL transcription reaction containing 5× transcription buffer (Thermo Fisher), 8 mM rNTPs, 1.25 units RiboLock RNase Inhibitor (Thermo Fisher), and 60 units of either T7 or SP6 RNA Polymerase (Thermo Fisher). After an overnight incubation at 30°C, 2 units RNA-free DNase (NEB) was added, and reactions left at 37°C for 30 min. RNA transcripts were precipitated by addition of LiCl and the resultant pellet washed in 70% ethanol before resuspension in RNase-free water. Capping of YFV and CHIKV replicon RNA transcripts was performed using the Vaccinia Capping System (NEB) according to manufacturer's recommendations. HEV and FCV transcripts were generated using the HiScribe T7 ARCA mRNA kit (NEB) using 1 µg of linearized plasmid template following manufacturer's instructions. The integrity of all RNAs was verified by running RNAs on a MOPS-formaldehyde agarose gel and visualizing product by SYBR Gold Staining (Thermo Fisher). When required, relative quantification of RNA species on the gels was achieved using a ChemiDoc XRS+ Imager (Bio-Rad).
Transcription buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Transcription buffer is a solution used to facilitate the in vitro transcription process. It provides the necessary ionic conditions and cofactors required for the efficient synthesis of RNA molecules by RNA polymerase enzymes.
Automatically generated - may contain errors
Lab products found in correlation
T7 rna polymerase, by Thermo Fisher Scientific (6 mentions)
Ribolock rnase inhibitor, by Thermo Fisher Scientific (4 mentions)
Rnasecure, by Thermo Fisher Scientific (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Sp6 rna polymerase, by Thermo Fisher Scientific (1 mentions)
Sybr gold staining, by Thermo Fisher Scientific (1 mentions)
Hiscribe t7 arca mrna kit, by New England Biolabs (1 mentions)
Vaccinia capping system, by New England Biolabs (1 mentions)
Chemidoc xrs imager, by Bio-Rad (1 mentions)
T7 polymerase, by Promega (1 mentions)
Rq1 dnase buffer, by Promega (1 mentions)
Spin x column, by Corning (1 mentions)
Rq1 dnase, by Promega (1 mentions)
Ribolock, by Thermo Fisher Scientific (1 mentions)
Nanodrop onec, by Thermo Fisher Scientific (1 mentions)
Fastdigest buffer, by Thermo Fisher Scientific (1 mentions)
Ecori, by Addgene (1 mentions)
Hindiii, by Addgene (1 mentions)
Hindiii, by Thermo Fisher Scientific (1 mentions)
Ecori, by Thermo Fisher Scientific (1 mentions)
8 protocols using transcription buffer
1
In Vitro RNA Transcription Protocol
2
In Vitro Transcription of Radiolabeled RNA
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The above oligonucleotides were annealed and added to a transcription reaction so that the final concentration was 0.1 μg/mL. Final concentrations of other components were 1× transcription buffer (Thermo Fisher), 10 mM DTT, 0.3 mM each ATP, GTP, and UTP, 0.5 pmol/ml alpha-32P CTP (3000Ci/mmol), 0.02 mM CTP, T7 RNA polymerase (Thermo Fisher). After 2 h incubation at 37°C, additional T7 polymerase was added and incubation continued for another 2 h. RQ1 DNase buffer was then added to 1× final concentration along with RQ1 DNase (Promega) and incubated at 37°C for 20 min. The RNA was ethanol precipitated and resuspended in TE. This was mixed 1:1 with formamide loading dye (93% formamide, 30 mM EDTA, 0.1× TBE, 0.5% bromophenol blue, 0.5% xylene cyanol), heated to 95°C for 5 min and loaded on a 8% acrylamide 7 M urea gel buffered with 1× TBE. After electrophoresis, bands were cut out of the gel and the RNA was extracted with 0.3 M sodium acetate at room temperature for 1 h. Samples were centrifuged briefly, liquid was removed, transferred to a Spin-X column (Corning), centrifuged, and ethanol precipitated. This procedure was then repeated. After ethanol precipitation, the pellet was resuspended in H2O.
3
Aptazyme Transcription and Purification
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Aptazyme constructs were amplified by Taq-PCR with primers inserting a T7 promotor to start transcription in vitro. The total volume of PCR reaction was 200 µl. PCR products were precipitated adding 1/10 volume of 3 M sodium acetate pH 5.7 and 3 volumes of 100% ethanol. Pelleted DNA was resuspended in 40 µl MilliQ and directly used for in vitro transcription [Thermo Scientific transcription buffer, 90 nM ATP, 2 mM CTP, 2 mM GTP and 2 mM UTP, 80 U RiboLock RNase Inhibitor (Thermo Scientific), 0.15 U of PPase (Thermo Scientific), 5 μCi 32P-α-ATP] and in presence of 25 μM blocking strand (5′-ATTTGGGACTCATCAGCTGG-3′). The blocking strand inhibits a formation of a catalytically active conformation of the ribozyme, thereby prevents self-cleavage in the presence of high magnesium concentrations needed for transcription. RNAs were purified by an 8% preparative PAGE gel.
4
In Vitro Transcription of RNA
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For in vitro transcription, 50 μl reactions containing 1 μg PCR products (generated using plasmid templates (Supplementary Table 7 ) and primers listed in Supplementary Table 6 ), 2 mM NTPs (ATP, UTP, GTP, CTP; ThermoFisher Scientific), T7 RNA polymerase (ThermoFisher Scientific), 1× transcription buffer (ThermoFisher Scientific) and 1 U/μl Ribolock (ThermoFisher Scientific) were incubated for 1 h at 37 °C. After transcription, samples were treated with Turbo DNase (ThermoFisher Scientific) and purified over Quick Spin RNA columns (Roche), according to the manufacturer’s instructions. The RNA concentration was determined using a Nanodrop Onec (ThermoFisher Scientific).
5
In Vitro Transcription of Plasmid DNA
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The template used for producing the DNA of the IGR and the control was pCF10 or pRS01, respectively (Table S2 ). A touchdown PCR was done with Phusion polymerase and a starting annealing temperature of 72°C with a decrease of 1°C/cycle. The PCR products were cloned into pRAV23 (Addgene) using EcoRI and HindIII restriction sites and transformed into Top10 cells. The cells were grown followed by miniprep (Qiagen) for plasmid isolation. The plasmids were digested with EcoRI (Thermo Fisher) and HindIII (Thermo Fisher) using the Fast Digest buffer (Thermo Fisher) and used as a template for T7 in vitro transcription. The T7 reaction mixture consisted of 9 mM MgCl2, 4 mM ribonucleotide triphosphates (rNTPs), 1× transcription buffer (Thermo Fisher), 2.5 to 5 μg DNA, and 0.05 mg/ml T7 RNA polymerase (Thermo Fisher) and in a 50-μl reaction volume, with incubation at 37°C for 1 h. The RNA was treated with proteinase K (Sigma) and DNase I (Sigma) according to manufacturer’s instructions. Afterward, the RNA was isolated by successive rounds of EtOH precipitation, and the dried pellet was resuspended in double-distilled water (ddH2O) at the desired concentrations.
6
Amplification and Transcription of GEX Libraries
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Fragmented GEX libraries were mixed with 25 μl of 2× Q5 master mix, 0.4 μl 100 µM oBW170 and 0.4 μl 100 µM oBW168, and amplified using the following protocol: 72 °C for 3 min, 98 °C for 30 s, 9 cycles of 98 °C for 10 s, 65 °C for 30 s, 72 °C for 30 s, a final incubation at 72 °C for 5 min and hold at 4 °C. Resulting samples were purified with a 1.2× SPRI reaction (elution volume, 40 μl) and converted into RNA by in vitro transcription. Briefly, 100 ng of amplified libraries were mixed with 8 μl 5× transcription buffer (Thermo Fisher, EP0112), 6 μl 2.5 mM rNTPs (NEB, N0466L), 1.5 μl of T7 RNA polymerase (Thermo Fisher, EP0112) and 1 μl of RNase-Out. Reactions were incubated at 37 °C for 2 h, after which DNA templates were digested with 3 μl DNase I (NEB, M0303L) and 3 μl 10× DNase I buffer (NEB, B0303S) at 37 °C for 15 min. RNA was purified using a 2× SPRI reaction (elution volume, 25 μl). These samples were the in vitro transcribed GEX libraries.
7
In Vitro Transcription of RNA
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Single-stranded DNA templates equipped with the complementary sequence of the T7 promoter at their 3′-ends were used for in vitro transcription. To generate the double-stranded T7 promoter region, an oligonucleotide representing the T7 promoter sequence (T7 primer) was annealed to its complementary sequence on the template. To prepare the partially ds oligonucleotide, 200 fmol of template and 25 pmol of T7 primer were mixed in a final volume of 5.5 µL containing 100 mM NaCl. The mixture was heated to 94°C for 30 sec, cooled down fast to 75°C, and then allowed to cool slowly to 27°C. To this mixture was added 8 µL H2O, 4 µL 5× Transcription buffer (Thermo Scientific), 1 µL 40 mM NTPs (Life Technologies), 0.5 µL RiboLock RNase Inhibitor (20 units) (Thermo Scientific), and 1 µL T7 RNA polymerase (20 units) (Thermo Scientific). The reaction and the negative control were incubated at 37°C for 2 h. Nucleic acids were phenol/chloroform extracted, ethanol precipitated, loaded on a 15% denaturing polyacrylamide gel, and in vitro transcribed RNA through the 2′,5′- and 3′,5′-branch was gel-purified.
8
Optimized RNA Extraction and Synthesis
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