Mmessage mmachine t7 ultra kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The MMESSAGE mMACHINE T7 Ultra Kit is a laboratory equipment product designed for in vitro transcription of RNA. It provides a system for efficient and reliable synthesis of high-quality RNA transcripts from DNA templates.

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342 protocols using mmessage mmachine t7 ultra kit

Generating mRNA Transcripts for Kdm3a, Kdm4b, and EGFP

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The coding region of Kdm3a was amplified from mouse testis complementary DNA using PCR with KOD-Plus-Neo DNA polymerase (Toyobo, Osaka, Japan). Forward and reverse primers contained T7 promoter and poly(T)₁₂₀ sequences, respectively. A step-down PCR amplification method was used, following the manufacturer’s instructions (Toyobo). Poly(A)-containing PCR products were subjected to in vitro transcription using a mMESSAGE mMACHINE T7 ULTRA Kit (Life Technologies, Carlsbad, CA, USA), following the manufacturer’s instructions. To generate a Kdm4b DNA template for in vitro transcription, pCMV-SPORT6 containing the full-length Kdm4b mRNA was used as the PCR template (DNAFORM, Kanagawa, Japan, Clone ID 3490671). Egfp cDNA was cloned using the pGEM-T Easy Vector System (Promega, Madison, WI, USA) and transcribed in vitro using the mMESSAGE mMACHINE T7 ULTRA Kit (Life Technologies) following the manufacturer’s instructions. The concentrations of the mRNAs were adjusted to 150 ng ml⁻¹ (Egfp), 550 ng ml⁻¹ (Kdm3a), or 450 ng ml⁻¹ (Kdm4b) to maintain a constant number of injected mRNA molecules. The primer sequences used for generating the templates for in vitro transcription are shown in Supplementary Table 9.

Fukuda A., Tomikawa J., Miura T., Hata K., Nakabayashi K., Eggan K., Akutsu H, & Umezawa A. (2014). The role of maternal-specific H3K9me3 modification in establishing imprinted X-chromosome inactivation and embryogenesis in mice. Nature Communications, 5, 5464.

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Cloning PPARGC1A 5' UTR for Luciferase Assay

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The full-length PPARGC1A 5′ UTR was cloned upstream of the nanoLuc coding sequence (Promega), with preservation of the endogenous PPARGC1A Kozak sequence. This sequence was then amplified using a forward PCR primer containing a T7 promoter sequence, and the resulting DNA was purified. RNA was transcribed and polyadenylated from this template using the mMESSAGE mMACHINE T7 Ultra kit (Invitrogen), checked for integrity by denaturing gel electrophoresis, purified by phenol-chloroform extraction and precipitation, and quantified by A₂₆₀ using a NanoDrop (Thermo Fisher).
For translation assays, RNA was transfected into COS cells using Lipofectamine 2000 in a ratio of 2.5 l Lipofectamine 2000 per 1 g RNA. COS cells were allowed to reach 90% confluence in 24-well plates prior to transfection. Immediately prior to transfection, medium was replaced with serum-free DMEM with or without sodium arsenite (Sigma). Cells were harvested 3 hr after transfection and luciferase activity was assessed using Nano-Glo luciferase assay (Promega) and a FLUOstar Omega plate reader (BMG Labtech).

Dumesic P.A., Egan D.F., Gut P., Tran M.T., Parisi A., Chatterjee N., Jedrychowski M., Paschini M., Kazak L., Wilensky S.E., Dou F., Bogoslavski D., Cartier J.A., Perrimon N., Kajimura S., Parikh S.M, & Spiegelman B.M. (2019). An evolutionarily conserved uORF regulates PGC1α and oxidative metabolism in mice, flies, and bluefin tuna. Cell metabolism, 30(1), 190-200.e6.

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Efficient Gene Editing using TALEN and CRISPR/Cas9

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RNAs for TALENs and CRISPR/Cas9 were prepared as described previously [5 (link)21 (link)27 (link)]. Briefly, TALEN and Cas9 mRNAs were synthesized in vitro from linear DNA templates (ToolGen, Korea) via the mMESSAGE mMACHINE T7 Ultra kit (Invitrogen, USA) according to the manufacturer's instructions. DNA templates for sgRNAs were synthesized in vitro by PCR. The sgRNAs were produced from these templates using a MEGA-shortscript T7 kit (Invitrogen, USA) according to the manufacturer's instructions. The sgRNAs were diluted in RNase-free injection buffer (0.25 mM EDTA, 10 mM Tris at pH 7.4) and introduced into the cytoplasm of fertilized eggs by microinjection. TALEN target sites were as follows: TALEN #1 for p16, 5′-TGCATGACGT GCGGGCACTG-3′; TALEN #2 for p16, 5′-TTCGGGG CGTTGGGCGAAAC-3′ for both B6 and FVB mice; TALEN #1 of p19, 5′-TTCGTGCGATCCCGGAGACC-3′; TALEN #2 of p19, 5′-TCACGAAAGCCAGAGCGC AG-3′ for both B6 and FVB mice. The following sequences were used for sgRNA synthesis: sgRNA #1 of p27, 5′-GCGGATGGACGCCAGACAAG-3′; sgRNA #2 of p27, 5′-GGACTTGGAGAAGCACTGCC-3′.

Park B.M., Roh J.I., Lee J, & Lee H.W. (2018). Generation of knockout mouse models of cyclin-dependent kinase inhibitors by engineered nuclease-mediated genome editing. Laboratory Animal Research, 34(4), 264-269.

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In Vitro Synthesis of TALEN Proteins

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The DNA fragments encoding the TALENs protein and an upstream T7 promoter were isolated by digesting the vectors with SacI and PmeI. The TALEN genes were transcribed in vitro, capped at 5′, and polyadenylated using an mMESSAGE mMACHINE T7 Ultra Kit (Invitrogen) according to the manufacturer's instructions. The mRNAs were translated in vitro using a Retic Lysate IVT Kit (Invitrogen) following the manufacturer's protocol.

Feng Y., Zhang S, & Huang X. (2014). A robust TALENs system for highly efficient mammalian genome editing. Scientific Reports, 4, 3632.

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In Vitro Transcription of CRISPR Components

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T7 promoter was added to the Cas9 coding region by PCR amplification using primer Cas9 F and R (Supplementary Table 6). The T7-Cas9 PCR product was gel-purified and used as the template for in vitro transcription using the mMESSAGE mMACHINE T7 ULTRA kit (Invitrogen). T7 promoter was added to the sgRNA template by PCR amplification using the primers shown in Supplementary Table 6. The T7-sgRNA PCR product was gel-purified and used as the template for in vitro transcription using the MEGAshortscript T7 kit (Invitrogen). Both the Cas9 mRNA and sgRNA were purified using the MEGAclear kit (Invitrogen) and eluted in RNase-free water.

Wu Y., Zhang J., Peng B., Tian D., Zhang D., Li Y., Feng X., Liu J., Li J., Zhang T., Liu X., Lu J., Chen B, & Wang S. (2019). Generating viable mice with heritable embryonically lethal mutations using the CRISPR-Cas9 system in two-cell embryos. Nature Communications, 10, 2883.

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Anesthetic-Assisted α-Bungarotoxin Labeling in Zebrafish

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Starting at 24 hpf, embryos were soaked in Danieau buffer containing 48 combinations of 0–200 μg/ml tricaine (Sigma Aldrich) and 0–0.003% v/v isoeugenol (Sigma Aldrich). A zebrafish codon optimized α-bungarotoxin ORF was synthesized (GeneArt/ Life Technologies) and sub-cloned into a construct with a T7 promoter at the 5’ end (pMTB-T7- α-bungarotoxin). The sequence is presented in the S1 Text and is available in GenBank (accession number KT279887). α-bungarotoxin mRNA was synthesized from a linearized plasmid using the mMessage mMachine T7 ULTRA kit (Invitrogen). Subsequently, mRNA was purified using RNAeasy Mini Kit (Qiagen). α-bungarotoxin protein was obtained from Tocris. 3 kDa dextran-Texas red and Alexa-Fluor 594 conjugated α-bungarotoxin were obtained from Invitrogen. 2.3 nl injections were performed using Nanoject II (Drummond).

Swinburne I.A., Mosaliganti K.R., Green A.A, & Megason S.G. (2015). Improved Long-Term Imaging of Embryos with Genetically Encoded α-Bungarotoxin. PLoS ONE, 10(8), e0134005.

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In Vitro Transcription of Cas9 and sgRNA

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T7 promoter was added to the N terminus of the Cas9/Cas9-msa/CasRX/Rad51 coding region by PCR amplification, using indicated primer (Additional file 4: Table S3). T7-Cas9/Cas9-msa/CasRX/Rad51 PCR production was purified and used as the template for in vitro transcription (IVT) using the mMESSAGE mMACHINE T7 ULTRA kit (Invitrogen, AM1345). T7 promoter was added to the sgRNA template by PCR amplification, using primers listed in Additional file 4: Table S3. The T7-sgRNA PCR product was purified and used as the template for IVT using the MEGA shortscript T7 kit (Invitrogen, AM1354). Both the mRNA and the sgRNAs were purified using a MEGA clear kit (Invitrogen, AM1908) and eluted in RNase-free water.

Chen H., Liu X., Li L., Tan Q., Li S., Li L., Li C., Fu J., Lu Y., Wang Y., Sun Y., Luo Z.G., Lu Z., Sun Q, & Liu Z. (2023). CATI: an efficient gene integration method for rodent and primate embryos by MMEJ suppression. Genome Biology, 24, 146.

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CRISPR-Cas9 Mediated Conditional Gene Editing

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The use and care of animals followed the guidelines of the Biomedical Research Ethics Committee of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. GOTI experiments were performed according to the previous method (33 (link)). Briefly, mRNA of A3G-BE5.13 or Cre was generated by attaching the T7 promoter to the coding region through PCR amplification and using its purified PCR product as the template for in vitro transcription (IVT) using the mMESSAGE mMACHINE T7 ULTRA Kit (Invitrogen). Similarly, for sgRNA, the T7 promoter was attached, and the MEGAshortscript T7 Transcription Kit (Invitrogen) was used for IVT. mRNA and sgRNA products were purified using the MEGAclear Transcription Clean-Up Kit (Invitrogen). Fertilized embryos were obtained from C57BL/6 females (4 weeks old) mated to heterozygous Ai9 males (JAX strain 007909). A3G-BE5.13 mRNA (50 ng/μl), Cre mRNA (2 ng/μl), and sgRNA (50 ng/μl) were mixed and injected using a FemtoJet microinjector (Eppendorf) into the cytoplasm of one blastomere of the two-cell embryo in a droplet of Hepes-CZB (Chatot-Ziomek-Bavister) medium containing cytochalasin B (5 μg/ml). The embryos were incubated at 37°C with 5% CO₂ under KSOM (Potassium simplex optimized medium) medium for 2 hours and transferred into oviducts of ICR (Institute for Cancer Research) females at 0.5 days post coitum.

Lee S., Ding N., Sun Y., Yuan T., Li J., Yuan Q., Liu L., Yang J., Wang Q., Kolomeisky A.B., Hilton I.B., Zuo E, & Gao X. (2020). Single C-to-T substitution using engineered APOBEC3G-nCas9 base editors with minimum genome- and transcriptome-wide off-target effects. Science Advances, 6(29), eaba1773.

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miRNA-133b Regulation of EGFP-tppp3 mRNA

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In vitro transcription of EGFP-tppp3 3′-UTR, EGFP-tppp3 mut-3′-UTR and mCherry mRNAs were performed with mMESSAGE mMACHINE T7 Ultra Kit (Invitrogen) and these synthesized mRNAs were purified with MEGAclearTM Kit (Invitrogen). Zebrafish embryos at one-cell stage were injected with a combing solution of sensor mRNA and mCherry mRNA. When applicable, 10 μM miR-133b duplex was added as an experimental group, while 10 μM non-sense duplex was added as a control. EGFP fluorescence was quantified at 24–28 h post-fertilization (hpf) using software Fiji-imageJ.

Huang R., Chen M., Yang L., Wagle M., Guo S, & Hu B. (2017). MicroRNA-133b Negatively Regulates Zebrafish Single Mauthner-Cell Axon Regeneration through Targeting tppp3 in Vivo. Frontiers in Molecular Neuroscience, 10, 375.

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Transcription of TnpB mRNA and ωRNA

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TnpB mRNA was transcribed using the mMESSAGE mMACHINE T7 Ultra Kit (Invitrogen, AM1345). T7 promoter was added to ωRNA template by PCR amplification of pCX2280 using forward and reverse primers in the supplementary files. The PCR products purified with Omega gel extraction Kit (Omega, D2500-02) as templates were transcribed using the MEGAshortscript Kit (Invitrogen, AM1354). The TnpB mRNA and ωRNA were purified by MEGAclear Kit (Invitrogen, AM1908), eluted with RNase-free water and stored at −80 °C.

Li Z., Guo R., Sun X., Li G., Shao Z., Huo X., Yang R., Liu X., Cao X., Zhang H., Zhang W., Zhang X., Ma S., Zhang M., Liu Y., Yao Y., Shi J., Yang H., Hu C., Zhou Y, & Xu C. (2024). Engineering a transposon-associated TnpB-ωRNA system for efficient gene editing and phenotypic correction of a tyrosinaemia mouse model. Nature Communications, 15, 831.

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