The plasmid pFK-Jc1 has been described recently and encodes the intragenotypic 2a/2a chimeric virus Jc1 (Pietschmann et al., 2006 (link)). In vitro transcripts of the individual constructs were generated by linearizing 5–10 μg of the Jc1 plasmid by digestion for 1 h with Mlu I. Plasmid DNA was extracted with phenol and chloroform and after precipitation with ethanol dissolved in RNase-free water. In vitro transcription reaction mixtures contained 80 mM HEPES (pH 7.5), 12 mM MgCl2, 2 mM spermidine, 40 mM dithiothreitol (DTT), a 3.125 mM concentration of each ribonucleoside triphosphate, 1 U of RNasin (Promega, Mannheim, Germany) per μL, 0.1 μg plasmid DNA/μL and 0.6 U of T7 RNA polymerase (Promega) per μL. After incubation for 2 h at 37°C, an additional 0.3 U of T7 RNA polymerase/μL reaction mixture was added, followed by another 2 h at 37°C. Transcription was terminated by the addition of 1.2 U of RNase-free DNase (Promega) per μg of plasmid DNA and 30 min incubation at 37°C. The RNA was extracted with acidic phenol and chloroform, precipitated with isopropanol and dissolved in RNase-free water. The concentration was determined by measurement of the optical density at 260 nm. Denaturing agarose gel electrophoresis was used to check RNA integrity.
T7 rna polymerase
Manufactured by Promega
Sourced in United States, Germany, Switzerland, China
T7 RNA polymerase is an enzyme that catalyzes the transcription of DNA into RNA. It recognizes and binds to the T7 promoter sequence, initiating the synthesis of RNA from the template DNA.
Automatically generated - may contain errors
Lab products found in correlation
Biotin rna labeling mix, by Roche (26 mentions)
Rneasy mini kit, by Qiagen (26 mentions)
Rnase free dnase 1, by Promega (23 mentions)
Rnasin, by Promega (16 mentions)
Streptavidin agarose bead, by Thermo Fisher Scientific (11 mentions)
Sp6 rna polymerase, by Promega (9 mentions)
Transcription buffer, by Promega (8 mentions)
Dnase, by Promega (7 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Rq1 dnase, by Promega (7 mentions)
Rnasin ribonuclease inhibitor, by Promega (6 mentions)
Rnase inhibitor, by Promega (5 mentions)
Dig rna labeling mix, by Roche (5 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Nanodrop, by Thermo Fisher Scientific (5 mentions)
Rnase free dnase, by Promega (4 mentions)
Turbo dnase, by Thermo Fisher Scientific (4 mentions)
Poly gel rna extraction kit, by Omega Bio-Tek (4 mentions)
Biotin rna labeling mix, by Merck Group (4 mentions)
Microspin g 25 column, by GE Healthcare (4 mentions)
259 protocols using t7 rna polymerase
1
Generating Jc1 Viral RNA Transcripts
2
Gene Expression Profiling by Whole Mount In Situ Hybridization
3
In vitro tRNA Transcription for M. tuberculosis
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tRNAs were obtained following in vitro transcription of PCR templates containing an integrated T7 RNA polymerase promoter sequence. Primers for M. tuberculosis tRNAs are given in Supplementary Table S1 . The T7 RNA polymerase in vitro transcription reactions were performed in 25 µl total volume with a 5 µl nucleotide mix of 2.5 mM NTPs (Promega). 50 to 100 ng of template were used per reaction with 1.5 µl rRNasin 40 U.ml−1 (Promega), 5 µl 5x optimized transcription buffer (Promega), 2 µl T7 RNA polymerase (20 U.ml−1) and 2.5 µl 100 mM DTT at 37 °C for 2 h25 (link). The tRNA products were isolated using trizol78 (link) and stored at a final concentration of 100 to 200 ng.µl−1, as monitored by NanoDrop.
4
Synthesis and Assembly of RNA Nanocubes
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The designing principles and production of RNA strands entering the composition of nanocubes functionalized with six siRNAs is comprehensively described elsewhere.24 The full list of RNA and DNA sequences used is available in Supporting Information . RNA molecules were purchased from Integrated DNA Technologies, Inc. or prepared by transcription of PCR amplified DNA templates; synthetic DNA molecules coding for the sequence of the designed RNA were purchased and amplified by PCR using primers containing the T7 RNA polymerase promoter. RNA molecules were prepared enzymatically by in vitro transcription using T7 RNA polymerase.65 (link) Transcription was performed in 50 mM Tris-HCl, pH 7.5; 2 mM spermidine; 1 mM DTT; 5 mM MgCl2; 0.5 mM MnCl2; 1 mM NTPs; 0.1 μM of DNA template; and T7 RNA polymerase (Promega). RNA, RNA–DNA and DNA–RNA nanocubes were assembled as detailed in Afonin et al.(17 (link),24 ) Cognate RNA–DNA hybrids were assembled as described in Afonin et al.(33 (link)) For the visualization of assembled nanoparticles, native-PAGE was used.66 (link),67 (link) Typically, assembly experiments were analyzed at 10 °C on 7% (29:1) native polyacrylamide gels in the presence of 89 mM Tris-borate, pH 8.3, and 2 mM Mg(OAc)2. A Hitachi FMBIO II Multi-View Imager was used to visualize SYBR Gold stained RNA-, RNA–DNA- and DNA–RNA-based nanoparticles.
5
Biotinylated RNA Riboprobe Synthesis from V. cholerae
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To construct the biotinylated RNA riboprobes, a DNA template was first prepared in the following PCR mixture: 200 μM deoxynucleoside triphosphates, 1 μM forward primer, 1 μM reverse primer, genomic DNA from V. cholerae strain JL2, 1× buffer, and Taq DNA polymerase (NEB). The DNA template was then used in an in vitro transcription assay performed with T7 RNA polymerase according to the manufacturer’s instructions: 0.5 mM each of ATP, CTP, and GTP; 0.3 mM UTP; 0.2 mM biotin-16-UTP; 10 μM dithiothreitol; DNA template; 1× buffer; and T7 RNA polymerase (Promega). The reaction mixture was allowed to incubate at 37°C for 1 to 3 h prior to addition of and incubation with RQ1 DNase at 37°C for 30 min. The riboprobe was purified using a Micro P-30 column (Bio-Rad).
6
In vitro Binding Assays for mCry1 5'UTR
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In vitro binding assays through UV crosslinking were performed as described previously [41 (link)]. In brief, XbaI-linearized pSK'-mCry1 5′UTR constructs were transcribed using T7 RNA polymerase (Promega) in the presence of [α-32P] UTP. 20 μg of whole cell extracts or 40 μg of cytosolic extracts were incubated with labeled RNAs at 30°C. After 30 min of incubation, the mixtures were UV-irradiated on ice for 15 min with a CL-1000 UV-crosslinker (UVP). The samples were detected with autoradiography after SDS-PAGE.
For Streptavidin-biotin RNA-affinity purification of mCry1 5′UTR-binding proteins, XbaI-linearized pSK'-mCry1 5′UTR constructs were transcribed using T7 RNA polymerase (Promega) in the presence of biotin-UTP. Cytoplasmic extracts prepared from NIH3T3 cells were incubated with or without biotinylated RNAs and subjected to streptavidin resin adsorption. For the competition assay, 2-fold molar excess of non-biotinylated competitor RNAs were additionally incubated. Resin-bound proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting.
For Streptavidin-biotin RNA-affinity purification of mCry1 5′UTR-binding proteins, XbaI-linearized pSK'-mCry1 5′UTR constructs were transcribed using T7 RNA polymerase (Promega) in the presence of biotin-UTP. Cytoplasmic extracts prepared from NIH3T3 cells were incubated with or without biotinylated RNAs and subjected to streptavidin resin adsorption. For the competition assay, 2-fold molar excess of non-biotinylated competitor RNAs were additionally incubated. Resin-bound proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting.
7
Protein-RNA Interaction Assay
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The interaction between KHDRBS3 and cDENND4C was tested by Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher) according to the manufacturer’s instruction. In brief, Biotin-labeled cDENND4C (bio-cDENND4C) were in vitro transcribed with the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Promega), treated with RNase-free DNase I (Promega) and purified with RNeasy Mini Kit (QIAGEN). The bio-cDENND4C and bio-antisense RNA (NC) were incubated with GECs lysates followed by extensive washes. Magnetic beads were added to prepare a probe–magnetic bead complex. The retrieved proteins were analyzed by Western blot with GAPDH as the control.
8
In Situ Hybridization of Protein-Coding Genes
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For the in situ hybridization experiments, we collected siliques with embryos at different stages from each genotype. To prepare probes, we amplified fragments of protein-coding genes by PCR from Ler cDNA and cloned them into pGEM-T Easy vectors (Promega, catalog no. A1360) for in vitro transcription using the Digoxigenin Labeling Kit (Roche, catalog no. 11277073910). Antisense and sense probes were synthesized using SP6 (Promega, catalog no. P1085) or T7 RNA polymerase (Promega, catalog no. P2075). The long probes were then hydrolyzed to an average length of 150 bp with carbonate buffer (2× buffer: 80 mM NaHCO3, 120 mM Na2CO3) and resuspended in 50% formamide at the desired concentration. The miR166 locked nucleic acid (LNA) probe (zma-miR166a) was obtained from the EXIQON company with 5′ end labeling of digoxigenin: /5′Dig/GGG GAA TGA AGC CTG GTC CGA. Primers for amplifying hybridization probes are listed in Supplemental Table 6 .
In situ hybridization experiments were performed as previously described (Zhang et al., 2017 (link)). For the miR166 LNA probe, the hybridization and washing were performed at 60°C and staining at 4°C, as previously described (Liu et al., 2009 (link)). For other probes, the hybridization and washing were performed at 55°C and staining at room temperature.
In situ hybridization experiments were performed as previously described (Zhang et al., 2017 (link)). For the miR166 LNA probe, the hybridization and washing were performed at 60°C and staining at 4°C, as previously described (Liu et al., 2009 (link)). For other probes, the hybridization and washing were performed at 55°C and staining at room temperature.
9
Gene Knockdown in S2 Cells
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For gene knockdown in S2 cells, specific dsRNAs were synthesized by in vitro transcription using T7 RNA polymerase (Promega). The siRNAs targeting human CASP3 were synthesized by RiboBio (Guangzhou, China). The primers used for dsRNA preparation and the sequences of siRNAs are shown in Table S1 .
10
Whole-mount chromogenic in situ hybridization
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For the sequence of all the primers used to amplify probes (relative to PDZ-RhoGEFs, Figure 7—figure supplement 2 ), see the Key Resources Table. Whole-mount chromogenic in situ hybridization was performed as described in Lancino et al., 2018 (link). Probes were synthetized using the manufacturer recommendation (T7 RNA polymerase, Promega, Cat# P2075, DIG-nucleotides, Jena Bioscence, Cat# NU-803-DIGX).
Images were captured with the Zeiss Axio ZOOM V16 microscope with the Zen Pro2 software, with a brightfield transmitted light optics. Post-processing steps were performed using the Extended-Depth Focus method to combine in focus regions from multiple z-planes and convert into in a transmitted light z-stack to generate a unique in-focus image.
Images were captured with the Zeiss Axio ZOOM V16 microscope with the Zen Pro2 software, with a brightfield transmitted light optics. Post-processing steps were performed using the Extended-Depth Focus method to combine in focus regions from multiple z-planes and convert into in a transmitted light z-stack to generate a unique in-focus image.
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