Maxiscript kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

The MAXIscript kit is a reagent system designed for in vitro transcription. It provides the necessary components to synthesize high-quality, radiolabeled or unlabeled RNA from DNA templates.

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Lab products found in correlation

Pierce magnetic rna protein pull down kit, by Thermo Fisher Scientific (8 mentions) Tnt coupled reticulocyte lysate system with sp6 rna polymerase, by Promega (4 mentions) α 32p utp, by PerkinElmer (4 mentions) 5600 plus, by AB Sciex (3 mentions) Pgem t easy vector, by Promega (3 mentions) Fish tag rna multicolor kit, by Thermo Fisher Scientific (3 mentions) Proteinase k, by Roche (2 mentions) 5 bromo 4 chloro 3 indolyl phosphate, by Roche (2 mentions) Anti digoxigenin antibody conjugated with alkaline phosphatase, by Roche (2 mentions) Monolith nt 115 instrument, by NanoTemper (2 mentions) Northern max gly kit, by Thermo Fisher Scientific (2 mentions) Rneasy minelute cleanup kit, by Qiagen (2 mentions) Ab94010, by Abcam (2 mentions) Anti flag affinity resin, by Merck Group (2 mentions) Flag peptide, by Merck Group (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Amersham hyperfilm mp, by GE Healthcare (2 mentions) Urea 6 polyacrylamide tbe gel, by Thermo Fisher Scientific (2 mentions) Dynamarker small rna plus, by Kodak (2 mentions) X ray film, by Kodak (2 mentions)

Close spelling variants of this product

MAXIscript SP6 Transcription Kit Ambion Maxiscript-T7 In Vitro Transcription Kit MAXIscript T7 Transcription Kit Ambion MAXIscript SP6/T7 In Vitro Transcription Kit MAXIscript in vitro transcription kit Ambion MAXIscript T7 Kit MAXIscript transcription kit MAXIscript® SP6 Kit Ambion MAXIscript Kit Maxiscript SP6/T7 kit

77 protocols using maxiscript kit

RNA Extraction and Blot Analysis of Viroids and mRNAs

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Total RNA was extracted from Agrobacterium-infiltrated N. benthamiana or viroid inoculated S. tuberosum leaf tissues using the hot-phenol method as previously described13 (link). For high molecular weight RNA gel blots, 15 μg of total RNA was separated on 1.2% agarose gels containing 6% formaldehyde and transferred to nylon N⁺ membrane. For mature PSTVd and siPSTVd detection, the [a-³²P]-UTP-labelled T7 RNA transcripts from cDNA clones in pGEM-Teasy vector using the MAXIscriptkit (Ambion) were used as probes; for GFP mRNA detection, the full length GFP were radioactively labelled by [a-³²P]-dCTP using a Rediprime™II Random Prime Labelling System(GE healthcare), for siGFP detection, the [a-³²P]-UTP-labelled T7 RNA transcripts from pGEM-Teasy-GFP vector using the MAXIscriptkit (Ambion) were used as probes; for Virp1 mRNA detection, the full length NbVirp1 were radioactively labelled by [a-³²P]-dCTP using a Rediprime™II Random Prime Labelling System(GE healthcare); for miR167, miR159 and U6 detection, [r-³²P]ATP-labeled specific oligonucleotide probe sequences were used.
Dot-blot hybridization detection of PSTVd accumulation was performed as described (Monger WA et al. 2015). The PSTVd DNA fragment was labeled using the DIG DNA PCR labeling kit (Mylab Corporation).

Lv D.Q., Liu S.W., Zhao J.H., Zhou B.J., Wang S.P., Guo H.S, & Fang Y.Y. (2016). Replication of a pathogenic non-coding RNA increases DNA methylation in plants associated with a bromodomain-containing viroid-binding protein. Scientific Reports, 6, 35751.

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Evaluating EV71 IRES Activity in SF268 Cells

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The bicistronic reporter plasmid pRHF-EV71-5′UTR, which contains the EV71 IRES between Renilla and Firefly luciferase open reading frames^{13 (link)} was used as the template for synthesis of capped reporter RNA using the MAXIscript kit (ThermoFisher). SF268 cells were seeded in 24-well plates. Two hundred ng of reporter RNA, 5 µl of SuperFect (Qiagen), and 400 µl of RPMI with 10% fetal calf serum were combined and added to one well of cells. Cells were incubated at 37 °C for 4 h, medium was changed, and various concentrations of DMA-135 were added. Two days after transfection, IRES activity was determined by measuring Renilla luciferase (RLuc) and Firefly luciferase (FLuc) activities using a dual-luciferase reporter assay system (Promega).

Davila-Calderon J., Patwardhan N.N., Chiu L.Y., Sugarman A., Cai Z., Penutmutchu S.R., Li M.L., Brewer G., Hargrove A.E, & Tolbert B.S. (2020). IRES-targeting small molecule inhibits enterovirus 71 replication via allosteric stabilization of a ternary complex. Nature Communications, 11, 4775.

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In Situ Hybridization of Bioengineered Tooth Germs

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Digoxigenin-labelled RNA probes were prepared with the MAXIscript Kit (Thermo Fisher Scientific, Waltham, MA, USA), in accordance with the manufacturer’s instructions. The DNA fragment of Fgf4 (NM_010202, located between 117 and 731) was amplified by polymerase chain reaction (PCR), using primers shown in Supplementary Table S1. The fragment was subcloned into the pGEM-T vector (Promega, Madison, WI, USA) and used to generate sense and antisense probes. Bioengineered tooth germs after 5-day organ culture were fixed in 4% PFA overnight at 4 °C, treated with 6% H₂O₂ and 0.1% Tween 20 in PBS at room temperature, and incubated with 10 µg/mL proteinase K (Roche, Mannheim, Germany) for 3 min at 30 °C. After post-fixation and prehybridization, samples were hybridized overnight at 70 °C with digoxigenin-labelled RNA probes. Samples were blocked with 10% normal sheep serum (Sigma) and then incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche), overnight at 4 °C. Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche) were used for signal detection.

Oyanagi T., Takeshita N., Hara M., Ikeda E., Chida T., Seki D., Yoshida M., Seiryu M., Takano I., Kimura S., Oshima M., Tsuji T, & Takano-Yamamoto T. (2019). Insulin-like growth factor 1 modulates bioengineered tooth morphogenesis. Scientific Reports, 9, 368.

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CRISPR-Cas9 Editing of Zebrafish ogg1 Gene

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The gRNA sequence was designed based on the information obtained from the CHOPCHOP website (http://chopchop.cbu.uib.no/). The target site located in exon 3 of the zebrafish ogg1 gene (5′-GCGAGACCGCAGATAGTCAC-3′) has 20 bases upstream of the protospacer adjacent motif (PAM) (TGG). The gRNA and the Cas9 mRNA were synthesized by in vitro transcription using the mMESSAGE mMACHINE T7 kit and the MAXIscript kit (both from Ambion; Thermo Fisher Scientific, Inc.), respectively, which were mixed in proportion (gRNA 50 ng/µl and Cas9 mRNA 150 ng/µl) and injected into one cell-phase zebrafish embryo (F0) (21 (link)). A total of 10-15 embryos were selected at 2 days post-fertilization (dpf) to extract the genomic DNA for sequencing and identification. Mature F0 zebrafish were then hybridized with wild-type AB zebrafish to produce the offspring (F1) containing heterozygous mutants, and the heterozygous F1 generations were then genotyped by tail fin-cutting sequencing to determine the presence of frameshift mutation. The F1 male and female zebrafish with the same mutant type were mated to produce the homozygous F2 mutant. The cataract model was constructed monocularly when the F2 mutant zebrafish grew to 3 months of age. The Cas9 template plasmid and gRNA template plasmid were donated by the Jiangsu Key Laboratory of Neurodegeneration.

Zhou T., Zhang J., Qin B., Xu H., Zhang S, & Guan H. (2020). Long non-coding RNA NONHSAT143692.2 is involved in oxidative DNA damage repair in the lens by regulating the miR-4728-5p/OGG1 axis. International Journal of Molecular Medicine, 46(5), 1838-1848.

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Identifying CD27-AS1-208 RNA-binding Proteins

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RNA pull-down assays were performed as previously described (29 (link)). In brief, LncRNA CD27-AS1-208 and negative control lncRNA CD27-AS1-208 antisense were transcribed in vitro using MAXIscript™ Kit (ThermoFisher, Waltham, MA) according to manufacturer’s instructions. Then, approximately 50 pmol biotinylated RNA from the previous step was added to 200 μg whole-cell lysate from A2058 cells to examine the protein binding to CD27-AS1-208 using Pierce™ Magnetic RNA-Protein Pull-Down Kit (ThermoFisher, Waltham, MA). The RNA-binding protein complexes were washed, eluted and analyzed by Mass spectrometry (MS) (5600-plus, AB SCIEX, MA).

Ma J., Shi Q., Guo S., Xu P., Yi X., Yang Y., Zhang W., Liu Y., Liu L., Yue Q., Zhao T., Gao T., Guo W, & Li C. (2022). Long Non-Coding RNA CD27-AS1-208 Facilitates Melanoma Progression by Activating STAT3 Pathway. Frontiers in Oncology, 11, 818178.

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Transcriptome Analysis in Mouse Tissues

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Total RNA (30 µg each) was isolated from mouse testes and other tissues, and subjected to northern blot analysis. DNA templates containing T7 or T3 RNA polymerase promoter site were
generated by PCR reactions with specific set of primers: forward primer, 5′-TAATACGACTCA CTATAGGGAGAATCTTCCTACGTACTCCCCTTTAGATGATC-3′ and reverse primer, 5′-AATTAACCCTCACTAAAGGGAGATCTAATCATTTATTATTCTCCAGCAGTCCAAGG-3′. Further, they were used for in vitro transcription using MAXIscript Kit (Thermo Fisher Scientific, Rochester, NY,
USA) to synthesize single-stranded digoxigenin (DIG)-labeled RNA probes according to the manufacturer’s protocol. Hybridization was performed using NorthernMax-Gly Kit (Thermo Fisher
Scientific) according to the manufacturer’s instructions.

LI C., SHEN C., SHANG X., TANG L., XIONG W., GE H., ZHANG H., LU S., SHEN Y., WANG J., FEI J, & WANG Z. (2019). Two novel testis-specific long noncoding RNAs produced by 1700121C10Rik are dispensable for male fertility in mice. The Journal of Reproduction and Development, 66(1), 57-65.

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In Situ Hybridization of Mouse Testes

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Adult mouse testes were fixed with Bouin’s buffer, embedded in paraffin, and sliced into 7 μm thick sections. DNA templates containing T7 or T3 RNA polymerase promoter site were generated by PCR reactions with specific set of primers as illustrated in Supplementary Table 2. They were then used for in vitro transcription using MAXIscript Kit (Thermo Fisher Scientific, USA) to synthesize single-stranded digoxigenin (DIG)-labeled RNA probes according to the manufacturer’s protocol. Hybridization was performed according to a previously reported protocol [47 ]. After hybridization and washing, the sections were incubated overnight with AP-coupled anti-DIG antibody at 4 °C. Color development reaction was performed at room temperature by incubating with BCIP/NBT substrate (Vector Laboratories, USA). After stopping the reaction, the sections were dehydrated, mounted, and then, observed under light microscope.

Li C., Shen C., Xiong W., Ge H., Shen Y., Chi J., Zhang H., Tang L., Lu S., Wang J., Fei J, & Wang Z. (2024). Spem2, a novel testis-enriched gene, is required for spermiogenesis and fertilization in mice. Cellular and Molecular Life Sciences: CMLS, 81(1), 108.

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Viral RNA oligonucleotide quantification protocol

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Viral RNA oligonucleotides as RNA standard was obtained by in-vitro-transcription from DNA using MAXIscript Kit (Thermo Scientific, UK), which comprised 562 bases of HTNV RNA targeted by the assay (22 (link)), and then quantified by an ultramicro microplate spectrophotometer (BioTek Epoch, USA), the concentration of the standard RNA stock was 800 ng/μL. For assessing viral load in each sample, the standard RNA was prepared with 10-fold serial dilution, ranging from 10¹⁰ copies to 10³ copies per reaction. Our standard RNA has a detection limit of 10¹⁰ copies to 10¹copies per reaction, and thus we set the threshold of detection for this assay at 100 genome copies per reaction. In-run analysis, standard curves were established with a slope of -3.6624, a Y-intercept of 41.68 cycles, and an R² value of 0.9996.

Ma R., Zhang X., Shu J., Liu Z., Sun W., Hou S., Lv Y., Ying Q., Wang F., Jin X., Liu R, & Wu X. (2021). Nlrc3 Knockout Mice Showed Renal Pathological Changes After HTNV Infection. Frontiers in Immunology, 12, 692509.

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RNA Gel Blot Analysis of PSTVd

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The detailed protocol has been reported previously [59 (link),60 (link)]. Briefly, after electrophoresis in 5% (w/v) polyacrylamide/8 M urea gel for 1 h at 200 V, RNAs were then transferred to Hybond-XL nylon membranes (Amersham Biosciences, Little Chalfont, United Kingdom) by a Bio-Rad semi-dry transfer cassette and were immobilized by a UV-crosslinker (UVP, Upland, CA). The RNAs were then detected by DIG-labeled UTP probes. PSTVd RNAs were prepared as described before [23 (link)]. SmaI-linearized pInt95-94(-) and pInt95-94(+) were used as templates for generating probes, using the MAXIscript kit (Thermo Fisher Scientific). The DIG-labeled probes were used for detecting PSTVd RNAs. RNA gel blot signals were obtained using ChemiDoc (Bio-Rad Laboratories) and quantified using ImageJ (https://imagej.nih.gov/ij/). The quantified signals with biological replicates were subject to graphing and statistical analysis using the built-in functions of Prism (GraphPad Software, LLC). The raw quantification data used for graphing were listed in S7 Dataset.

Dissanayaka Mudiyanselage S.D., Ma J., Pechan T., Pechanova O., Liu B, & Wang Y. (2022). A remodeled RNA polymerase II complex catalyzing viroid RNA-templated transcription. PLoS Pathogens, 18(9), e1010850.

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Measuring RNA-Protein Interactions by MST

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MST experiments were performed according to previous reports [34 (link),48 (link)]. A set of full-length ncRNAs containing WT or derivative OsiR for MST was transcribed in vitro by GenePharma using a MAXIscript kit (Thermo Fisher, MA, USA). Another set of 6-FAM-labeled 70-nt single-stranded RNAs (ssRNAs) containing WT katE2 mRNA (N- katE2 competitors) was synthesized by GenePharma (GenePharma, Jiangsu, China), as listed in Table S3. Four microliters of sample containing 250 nM labeled probe and increasing concentrations of a non-labeled competitor (from 10 nM to 340 µM) were loaded into standard treated silicon capillaries (Monolith NT.115 series capillaries; catalog no. MO-K002). Measurements were carried out using a Monolith NT.115 instrument (NanoTemper Technologies, Munich, Germany) at 25 °C in diethyl pyrocarbonate (DEPC)-treated water with 40% excitation power and medium MST-Power. The dissociation constant (K_d) was calculated as described previously [49 (link)]. Data analyses were performed using Nanotemper Analysis software (NanoTemper Technologies, Munich, Germany).

Gao L., Chen X., Tian Y., Yan Y., Zhan Y., Zhou Z., Zhang W., Lin M, & Chen M. (2020). The Novel ncRNA OsiR Positively Regulates Expression of katE2 and is Required for Oxidative Stress Tolerance in Deinococcus radiodurans. International Journal of Molecular Sciences, 21(9), 3200.

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