Maxiscript sp6 transcription kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The MAXIscript SP6 Transcription Kit is a lab equipment product designed for in vitro synthesis of SP6 RNA transcripts. It includes the necessary reagents for performing SP6 RNA transcription reactions.

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Lab products found in correlation

T7 rna polymerase, by Thermo Fisher Scientific (2 mentions) Megaclear transcription clean up kit, by Thermo Fisher Scientific (1 mentions) Pierce magnetic rna protein pull down kit, by Thermo Fisher Scientific (1 mentions) Quant it rna assay kit, by Thermo Fisher Scientific (1 mentions) Qubit fluorometer, by Thermo Fisher Scientific (1 mentions) Pcs2 plasmid, by Addgene (1 mentions) T4 dna ligase, by Thermo Fisher Scientific (1 mentions) T4 dna ligase, by Qiagen (1 mentions) Pegfp plasmid, by Addgene (1 mentions) Qiaquick gel extraction kit, by Qiagen (1 mentions)

Spelling variants of this product

MAXIscript kit (77 citations) MAXIscript (20 citations) MAXIscript SP6/T7 Transcription Kit (13 citations) SP6 transcription kit (10 citations) MAXIscript transcription kit (7 citations) MAXIscript™ SP6/T7 in vitro transcription kit (4 citations) MAXIscript® SP6 Kit (3 citations) Transcription Kits (2 citations) Ambion MAXIscript SP6/T7 In Vitro Transcription Kit (2 citations)

8 protocols using maxiscript sp6 transcription kit

Cloning and in vitro Transcription of Zebrafish and Mouse lncRNAs

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The cDNAs of zebrafish lmx1bb and zebrafish and mouse CNIB1 lncRNAs were cloned into pGEM-T easy vector (General Biosystems, China). The plasmids were linearized to produce DNA templates and RNAs were in vitro transcribed using T7 RNA polymerase (Invitrogen, Cat. No. 18033019) or MAXIscript SP6 Transcription Kit (Invitrogen, Cat. No. AM1330) in combination with normal NTPs (Invitrogen, Cat. No. 18109017). In vitro transcribed RNAs were further used in zebrafish for overexpression, northern blotting, or ISH assays directly or after biotin/digoxigenin modifications, as described below.

Wang F., Ren D., Liang X., Ke S., Zhang B., Hu B., Song X, & Wang X. (2019). A long noncoding RNA cluster-based genomic locus maintains proper development and visual function. Nucleic Acids Research, 47(12), 6315-6329.

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In Vitro Transcription of Synage mRNA

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The cDNA of mouse Synage (n424059) was cloned into pcDNA3.1 vector. The plasmid was linearized to produce a DNA template, and full-length sense or antisense RNAs were transcribed in vitro using T7 RNA polymerase (Invitrogen, 18033019) or MAXIscript SP6 Transcription Kit (Invitrogen, AM1330) in combination with biotinylated NTPs (Roche, 11685597910). In vitro transcribed RNAs were further used for FISH assays in cerebellar sections directly as described below, or for EMSAs as described above.

Wang F., Wang Q., Liu B., Mei L., Ma S., Wang S., Wang R., Zhang Y., Niu C., Xiong Z., Zheng Y., Zhang Z., Shi J, & Song X. (2021). The long noncoding RNA Synage regulates synapse stability and neuronal function in the cerebellum. Cell Death and Differentiation, 28(9), 2634-2650.

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Whole-mount in situ hybridization of zebrafish

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Whole-mount in situ hybridization (WISH) was performed as described previously (Wei et al, 2014 (link); Yu et al, 2019 (link)). All the templates of RNA probes were cloned from the cDNA library of wild-type embryo at 3 dpf and confirmed by Sanger sequencing. The digoxigenin-labeled RNA probes were synthesized using MAXIscript™ SP6 Transcription Kit (Invitrogen, United States). More than 48 zebrafish embryos were used to ensure that the proportion of homozygous embryos was above 12. WISH was conducted with three independent experiments. The images were captured using an optical microscope (BX53, Olympus). After imaging, DNA was extracted from the embryos for genotyping.

Han Y., Sun K., Yu S., Qin Y., Zhang Z., Luo J., Hu H., Dai L., Cui M., Jiang C., Liu F., Huang Y., Gao P., Chen X., Xin T., Ren X., Wu X., Song J., Wang Q., Tang Z., Chen J., Zhang H., Zhang X., Liu M, & Luo D. (2024). A Mettl16/m6A/mybl2b/Igf2bp1 axis ensures cell cycle progression of embryonic hematopoietic stem and progenitor cells. The EMBO Journal, 43(10), 1990-2014.

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HSATII RNA Transcription and Transfection

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HSATII containing fragment on chromosome 10 was amplified by PCR with M13 forward and reverse primers as previously described (38 (link)) and subjected to in vitro transcription with SP6 RNA polymerase following the MAXIscript SP6 Transcription Kit recommendations (Thermo Fisher Scientific, catalog AM1308). In vitro transcribed HSATII was purified with Megaclear Transcription Clean-Up Kit (Thermo Fisher Scientific, catalog AM1908). Each RNA (500 fmol) was then transfected using jetMESSENGER (Polyplus-transfection, catalog 15001) in accordance with the manufacturer’s instructions. After 48 hours, the cells were harvested and gene expression was analyzed by real-time quantitative PCR.

Porter R.L., Sun S., Flores M.N., Berzolla E., You E., Phillips I.E., KC N., Desai N., Tai E.C., Szabolcs A., Lang E.R., Pankaj A., Raabe M.J., Thapar V., Xu K.H., Nieman L.T., Rabe D.C., Kolin D.L., Stover E.H., Pepin D., Stott S.L., Deshpande V., Liu J.F., Solovyov A., Matulonis U.A., Greenbaum B.D, & Ting D.T. (2022). Satellite repeat RNA expression in epithelial ovarian cancer associates with a tumor-immunosuppressive phenotype. The Journal of Clinical Investigation, 132(16), e155931.

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RNA Pulldown Assay for CMTM6 Interactors

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RNA pulldown assay was performed with Pierce Magnetic RNA-Protein PullDown Kit (Thermo Fisher) following the manufacturer’s instructions. Based on luciferase reporter results, we designed three sequential RNA probes across 660–1259, 1260–1859 and 1860–2560 of CMTM6 transcript, respectively. RNA probes were obtained from corresponding cDNA fragments using MAXIscript SP6 Transcription Kit (Thermo Fisher). Pierce RNA 3′ End Desthiobiotinylation Kit was used to label RNA probes, and 50 pmol of labelled RNA was immobilized on 50 μl of streptavidin magnetic beads for 30 min at room temperature with agitation. Cell lysates were prepared in IP Lysis buffer and 50 μg of cell lysates were incubated with RNA probes for 1 h at 4 °C on rotator. Protein species were recovered by elution and analyzed by immunoblotting.

Liu Y., Li X., Zhang H., Zhang M, & Wei Y. (2021). HuR up-regulates cell surface PD-L1 via stabilizing CMTM6 transcript in cancer. Oncogene, 40(12), 2230-2242.

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Quantifying SHFV Viral RNA by RT-qPCR

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RNA in 200 μl of culture fluid harvested from infectious clone transfected MA104 monolayers was extracted with TRI Reagent. Reaction mixtures contained extracted RNA, the primer pair (1 μM) and probe (0.2 μM) in a final volume of 10 μl. Primer/probe sequences targeted either the nucleocapsid (forward primer 5'-tcccacctcagcacacatca-3', TaqMan probe 5′-6FAMaacagctgctgatcaggtMGBNFQ-3′ and reverse primer 5′-ccgcctccgttgtcgtagt-3′) or helicase (nsp9) (forward primer 5′-cgtacacccgccgtctgct-3′, TaqMan probe 5′-6FAMttgacgttctcacaaaggMGBNFQ-3′, and reverse primer 5′-cggcaagtggcatccaa-3′) regions. Quantitative real time RT-PCR was performed and the data were analyzed as previously described (Scherbik et al., 2006 (link)). Extracellular SHFV genomic RNA was quantified using a standard curve generated with serial dilutions of a known concentration of SHFV RNA that had been in vitro transcribed with a MAXIscript SP6 transcription kit (Ambion) from either the nucleocapsid or nsp9 gene region of the SHFVic cDNA, digested with DNase, extracted with phenol-chloroform, precipitated with ethanol and then quantified by UV spectrophotometry.

Vatter H.A., Di H., Donaldson E.F., Baric R.S, & Brinton M.A. (2014). Each of the eight simian hemorrhagic fever virus minor structural proteins is functionally important. Virology, 0, 351-362.

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In vitro RNA Transcription and Electroporation

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2 μg of plasmid DNA were linearized with SmaI (New England Biolabs) and used as template for in vitro transcription with MAXIscript SP6 transcription kit (Ambion, Huntingdon, United Kingdom). The amount of RNA was determined by using Quant-iT RNA assay kit and Qubit-Fluorometer (Invitrogen). RNA quality was verified by agarose gel electrophoresis. 1 μg RNA was used for electroporation of 3 x 10⁶ cells. Electroporation of RNAs in MDBK cells was carried out as described previously [71 (link)]. After electroporation, cells were transferred into complete MEM and seeded as required for the assay.

Dubrau D., Tortorici M.A., Rey F.A, & Tautz N. (2017). A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis. PLoS Pathogens, 13(2), e1006134.

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Generating GFP-snap29 Plasmid for snap29K164* Mutant Rescue

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To generate the GFP-snap29 plasmid used for snap29^K164* mutant rescue, the zebrafish snap29 coding sequence was amplified using as template 24 hpf embryo cDNA and as primers BglII-Snap29 forward 5′-TCGAGAAGATCTATGTCTGCCTACCCCAAATCCC-3′ and XhoI-Snap29 reverse 5′-ATCGCCCTCGAGCTATTTAAGGCTTTTGAGCTG-3′. Both the PCR product and the pEGFP plasmid (Addgene) were digested using BglII and XhoI restriction enzymes (New England Biolab, NEB), purified using QIAquick Gel Extraction Kit protocol (QIAGEN) and subjected to ligation with T4 DNA ligase (NEB) according with manufacturer instructions. The plasmid obtained was used as a template for a second PCR using as primers BamHI-GFP forward 5′-ATCGCGGGATCCATGTGAGCAAGGGCGAGG-3′ and XhoI-Snap29 reverse. Both PCR product and pCS2 plasmid (Addgene) were digested with BamHI and XhoI restriction enzymes, purified and subjected to ligation with T4 DNA ligase (NEB).
pCS2 GFP-snap29 was used as templates to synthesize mRNAs using MAXIscript SP6 Transcription Kit (Ambion). 200 pg of mRNA were injected in one-cell stage embryos.

Mastrodonato V., Beznoussenko G., Mironov A., Ferrari L., Deflorian G, & Vaccari T. (2019). A genetic model of CEDNIK syndrome in zebrafish highlights the role of the SNARE protein Snap29 in neuromotor and epidermal development. Scientific Reports, 9, 1211.

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