Transcription buffer

Manufactured by Promega

Sourced in Singapore, United States

Transcription buffer is a solution formulated to facilitate the in vitro transcription of DNA into RNA. It provides the necessary ionic conditions and chemical components required for the optimal activity of RNA polymerase enzymes.

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T7 transcription buffer (3 citations) 5x Transcription optimized buffer (2 citations)

12 protocols using transcription buffer

Mechanism of AID-mediated DNA Mutagenesis

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The 570 bp substrates for wild-type (WT) E2A, mut1, and mut2 containing the T7 promoter were generated by digestion of pDL14, pDL31, and pDL32, respectively, with BlpI and XhoI at 37°C for 2 hours in 1× CutSmart buffer followed by native PAGE (5%) purification. The recovered DNA was quantified by real-time qPCR with DL127, DL128, and DL129 (45 cycles of 94°C 30 s, 65°C 1 min).
The AID reactions containing 40 fmol/µl of GST-AID, 250 µM rNTP mix, 10 mM DTT, and 2 units/µl of T7 RNA polymerase in 1 × transcription buffer (Promega), with 5 fmol/µl of the 570 bp DNA substrate added last to initiate the transcription reaction, were incubated at 37°C for 60 min. T7 RNA polymerase was omitted for the untranscribed reactions. Where indicated, 50 ng/µl of RNase A was added at the same time as other components to the reactions. The GST-AID variant of human AID with a carboxy (C) terminal hexa-His-tag is a generous gift from Dr. Myron Goodman (Pham et al., 2019 (link)). Reactions were terminated and purified using HighPrep PCR system (MAGBIO) and then digestion with HaeII restriction enzyme at 37°C for 2 hours was done followed by another purification with HighPrep PCR system. HaeII digestion of the reaction generated a 212 bp E2A substrate containing the 23 bp E2A fragile zone which was used as template in the Maximum-Depth Sequencing (MDS) library construction described below.

Liu D., Eddie Loh Y.H., Hsieh C.L, & Lieber M.R. (2021). Mechanistic basis for chromosomal translocations at the E2A gene and its broader relevance to human B cell malignancies. Cell reports, 36(2), 109387.

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CRISPR Guide RNA Design and Delivery

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CRISPR target sites were selected based on their proximity to the start and stop codons of the coding sequence of the targeted gene, and also by the requirement for a protospacer adjacent motif (PAM) sequence (NGG) at the 3′ end of target site. We created and annealed oligos containing a T7 promoter sequence, the target sequence, and an additional constant region to create the template for the guide RNAs (Additional file 1). These templates were transcribed in vitro using T7 RNA polymerase (Promega) in a reaction containing transcription buffer (Promega), RNase inhibitor (Promega), and rNTPs. A linearized plasmid encoding cas9 [26 (link)] was also transcribed in vitro using the Sp6 mMessage mMachine Kit (Ambion). The two guide RNAs targeting each gene were combined with cas9 mRNA and phenol red, and 1-2 nl of this mixture was injected into the cell of early 1-cell stage embryos.

Maurer J.M, & Sagerström C.G. (2018). A parental requirement for dual-specificity phosphatase 6 in zebrafish. BMC Developmental Biology, 18, 6.

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Synthesis and Validation of Ltbp3 Riboprobe

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The Ltbp3 probe was synthesized from PCR-generated DNA templates kindly provided by the EURExpress consortium (http://www.eurexpress.org). The template sequence is given in Supplementary Material, Fig. S3. DIG-labeled antisense riboprobe was transcribed in vitro by incubation for 2 h at 37°C using 1 µg of the PCR product, 20 U SP6 RNA polymerase, 5× transcription buffer (Promega), 10× DIG RNA labeling Mix (Roche), 0.5 M 1,4-Dithiothreitol, 20 U RNAse inhibitor (Roche) in a 20-µl volume. The reaction was stopped with 2 µl EDTA (0.2 m, pH 8), and RNA was precipitated with 1 µl yeast tRNA (10 mg/ml), 2.5 µl LiCl (4 M) and 75 µl absolute ethanol, followed by an incubation for 30 min at −80°C and centrifugation at 12 000 rpm (30 min at 4°C). The pellet was washed with 0.5 ml ethanol (70%) and re-centrifuged. The supernatant was discarded and the pellet was allowed to dry. The probe was then diluted in 20 µl sterile H₂O. The quality of the probe was verified by electrophoresis in a 1% agarose gel. If no smear was observed and the size was as expected, the probe was considered to be ready for use. The quantity of RNA was evaluated by Nanodrop (ND-1000 Spectrophotometer, Labtech) and adjusted to 150 ng/µl in hybridization buffer, then stored at −20°C until use.

Huckert M., Stoetzel C., Morkmued S., Laugel-Haushalter V., Geoffroy V., Muller J., Clauss F., Prasad M.K., Obry F., Raymond J.L., Switala M., Alembik Y., Soskin S., Mathieu E., Hemmerlé J., Weickert J.L., Dabovic B.B., Rifkin D.B., Dheedene A., Boudin E., Caluseriu O., Cholette M.C., Mcleod R., Antequera R., Gellé M.P., Coeuriot J.L., Jacquelin L.F., Bailleul-Forestier I., Manière M.C., Van Hul W., Bertola D., Dollé P., Verloes A., Mortier G., Dollfus H, & Bloch-Zupan A. (2015). Mutations in the latent TGF-beta binding protein 3 (LTBP3) gene cause brachyolmia with amelogenesis imperfecta. Human Molecular Genetics, 24(11), 3038-3049.

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Mechanism of AID-mediated DNA Mutagenesis

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In Vitro mRNA Synthesis Protocol

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Linearized plasmid (2.5 μg) was added to a mixture containing transcription buffer (Promega), 10 mM DTT, 2.5 mM cap analog, 50 units RNasin, 200 units RNA polymerase, and 0.5 mM each UTP, ATP, CTP, GTP in a total volume of 50 uL. The reaction was incubated at 37 °C for 90 min, followed by the addition of another 200 units of polymerase and incubation for 90 additional minutes. The reaction mixture was extracted twice with phenol pH 4.5 and twice with 24:1 chloroform: isoamylalcohol. The mRNA was precipitated with ethanol, suspended in 50 ul of H₂O and run though a NucAway spin column (Ambion) to remove unincorporated nucleotides. mRNA concentrations were determined using a NanoDrop spectrometer (Thermo Scientific), and samples were stored at − 80 °C until use.

Bertke M.M., Dubiak K.M., Cronin L., Zeng E, & Huber P.W. (2019). A deficiency in SUMOylation activity disrupts multiple pathways leading to neural tube and heart defects in Xenopus embryos. BMC Genomics, 20, 386.

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Radiolabeling of RNA Transcripts

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³²P-Labeled RNAs were transcribed in a 10 μl volume containing 20 U of T7 RNA polymerase (Promega), Transcription Buffer (Promega), 1 mM DTT (Promega), 12 U of RNasin Plus (Promega), NTP mixture (0.5 mM ATP, 0.5 mM CTP, 0.1 mM UTP and 0.1 mM GTP), 1 μg of DNA template, 1 mM m⁷G(5′)ppp(5′)G RNA Cap Structure Analog (New England Biolabs) and 2.96 TBq/mmol [α-³²P]UTP (Perkin Elmer). Cap Structure Analog was not added for U6 snRNA or unlabeled 700 nt RNA. After a 60 min incubation at 37°C, RNA was recovered from the supernatants by phenol/chloroform extraction and purified using G-50 microcolumns (GE Healthcare). RNA was then precipitated with ethanol and dissolved in H₂O.

Dantsuji S., Ohno M, & Taniguchi I. (2023). The hnRNP C tetramer binds to CBC on mRNA and impedes PHAX recruitment for the classification of RNA polymerase II transcripts. Nucleic Acids Research, 51(3), 1393-1408.

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In Vitro Transcription of β-Globin

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In vitro transcription was performed using 0.5 μg/μL linear β-Globin DNA in a reaction buffer containing 0.67 μM (α-³²P) UTP, 0.4 mM ATP, 0.4 mM CTP, 0.1 mM UTP, 0.1 mM GTP, 2 mM m7G(5′)ppp(5′G) (cap analog), 2 mM DTT, 10 U/μL ribonuclease inhibitor, 7.5 U/μL T7 RNA polymerase (Promega) and 1× transcription buffer (Promega). Reactions were incubated at 37°C for 2 h, gel purified using denaturing PAGE, eluted from the gel (elution buffer: 0.5 mM NaOAc, pH 5.6, 0.1% SDS, 10 mM Tris, pH 7.5, 1 mM EDTA), ethanol precipitated, and resuspended in nuclease free water.

Aubol B.E., Wu G., Keshwani M.M., Movassat M., Fattet L., Hertel K.J., Fu X.D, & Adams J.A. (2016). Release of SR Proteins from CLK1 by SRPK1: A Symbiotic Kinase System for Phosphorylation Control of Pre-mRNA Splicing. Molecular cell, 63(2), 218-228.

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Evaluating Angiotensin 1-7 Effects on Transcription

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A standard in vitro RNA transcription system (Promega, Madison, WI, USA) was used to study the effects of Ang 1-7 on mRNA transcription in isolated nuclei. Freshly isolated nuclei (100 μg) were pretreated with Ang 1-7 (1 μM) alone or combined with the Mas receptor antagonist A779 (10 μM) for 30 min at 37 °C. Pre-treated nuclei were then incubated with an in vitro RNA transcription system consisting of 500 μM of ATP, GTP, and UTP; and 2 U/μl RNasin in transcription buffer (Promega) at 37 °C for 1 h. After treatments, RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s instructions. RNA (1.8 μg) was reverse transcribed to cDNA with deoxynucleotide triphosphate (dNTP), random primers, and Moloney murine leukemia virus reverse transcriptase (MMLV; 200 U; Invitrogen). In order to evaluate the expression of AT1, AT2, and MasR genes, RT-PCR was performed as described previously.

Costa-Besada M.A., Valenzuela R., Garrido-Gil P., Villar-Cheda B., Parga J.A., Lanciego J.L, & Labandeira-Garcia J.L. (2017). Paracrine and Intracrine Angiotensin 1-7/Mas Receptor Axis in the Substantia Nigra of Rodents, Monkeys, and Humans. Molecular Neurobiology, 55(7), 5847-5867.

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Probing snoRNA-K-Ras Protein Interactions

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For wild-type and mutant snoRNAs, pCR2.1TOPO vector containing snoRNAs under the control of the SP6 promoter and flanked with an SspI site at the 3′ end was used. Plasmid DNA was digested with SspI, and 4 pg of linear DNA was then in vitro transcribed in a 50-µl total volume consisting of 1× Transcription buffer (Promega), 10 mM DTT (Promega), 1 mM NTPs (Invitrogen), 40 U RNaseOUT (Invitrogen) and 60 U SP6 RNA polymerase at 37 °C for 4 h, after which DNA was digested by the addition of 2 U DNase I at 37 °C for 15 min. For gel-shift assays, 0.2 µM of the full-length or deletion-mutant forms of snoRNAs SNORD50A, SNORD50B or scrambled 74-bp (USCR) RNAs was used with the indicated amount or 0.6 µM of purified recombinant K-Ras protein (Abcam, ab96817). snoRNAs were denatured at 90 °C for 30 s in 10 µl of water, and samples were immediately transferred to ice; after adding an equal volume of 40 mM HEPES (pH 7.4) with 2 mM DTT, the sample volume was brought up to room temperature for 5 min to allow equilibration. Next, K-Ras protein was added, and samples were incubated an additional 15 min and then analyzed using a 10% TBE polyacrylamide gel; gels were scanned using a Typhoon 9410 scanner (GE Healthcare), with band intensity quantified using ImageJ software.

Siprashvili Z., Webster D.E., Johnston D., Shenoy R.M., Ungewickell A.J., Bhaduri A., Flockhart R., Zarnegar B.J., Che Y., Meschi F., Puglisi J.D, & Khavari P.A. (2015). The noncoding RNAs SNORD50A and SNORD50B bind K-Ras and are recurrently deleted in human cancer. Nature genetics, 48(1), 53-58.

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In Vitro Transcription and RNA Hybridization

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EcoRI-linearized FHV_T_Rluc and FHV_T_minus and XhoI-linearized pEAV221Δ [34 (link)] plasmids were used as templates for in vitro transcription. Transcripts of FHV_T_Rluc and FHV_T_minus corresponding to capture probes for minus and plus strands, respectively, and a transcript containing nucleotides 1-2042 of equine arteritis virus (EAV) genome were prepared using mMESSAGE mMACHINE T7 Transcription Kit (Ambion) according to the manufacturer’s instructions. In vitro transcription reactions to prepare ³²P-labeled transcripts of FHV_T_Rluc and FHV_T_minus consisted of 750 ng of the respective linearized plasmid, transcription buffer (Promega), 800 U/mL T7 RNA polymerase (Promega), 1000 U/mL RiboLock (Thermo Fisher Scientific), 5 mM DTT, 1 mM ATP, UTP, GTP and CTP, and 0.133 μM α-³²P-CTP (20 μCi) (Perkin Elmer, Ayer Rajah, Singapore). The reactions were incubated for 2 h at 37 °C followed by RQ1 RNase-free DNase treatment (40 U/mL; Promega) for 35 min at 37 °C and inactivation by RQ1 DNase STOP solution (Promega) for 10 min at 65 °C. Unincorporated label was removed using RNase-free Micro Bio-Spin P-30 Gel Columns (Bio-Rad). Hybridization with the ³²P-labeled RNA transcripts and IVRA products was performed as described [34 (link)].

Quirin T., Chen Y., Pietilä M.K., Guo D, & Ahola T. (2018). The RNA Capping Enzyme Domain in Protein A is Essential for Flock House Virus Replication. Viruses, 10(9), 483.

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