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Maxiscript sp6 t7 in vitro transcription kit

Manufactured by Thermo Fisher Scientific
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The MAXIscript™ SP6/T7 in vitro transcription kit is a tool used for the production of RNA transcripts from DNA templates. It contains the necessary components, including RNA polymerases, nucleotides, and buffers, to enable the in vitro synthesis of high-quality RNA from SP6 or T7 promoter-driven DNA templates.

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Lab products found in correlation

Dig rna labeling mix, by Roche (1 mentions) Pgem t easy vector, by Promega (1 mentions) Dig rna labelling mix, by Roche (1 mentions) Cm1850, by Leica (1 mentions) Digoxigenin dig 11 utp, by Roche (1 mentions) Dig nucleic acid detection kit, by Roche (1 mentions) Hindiii, by New England Biolabs (1 mentions) Anti dig ap antibody, by Roche (1 mentions) Ecori, by New England Biolabs (1 mentions)

Close spelling variants of this product

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

4 protocols using maxiscript sp6 t7 in vitro transcription kit

1

Synthesis of NGF mRNA Probes

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mRNA probes to identify NGF mRNA receptors were synthetized by in vitro transcription (IVT) using the MAXIscript™ SP6/T7 in vitro transcription kit (Catalogue number AM1312, Invitrogen by Thermo Fisher Scientific, Carlsbad, CA. USA) and following the manufacturer’s instructions. One microgram of the DNA template was transcribed to RNA in 20 µL volume reaction, using primers associated with the T7 promoter sequence (NTRK1/NTRKA forward 5′-ATGGATGGAAACCCTGAGCC-3′; NTRK1/NTRKA T7 reverse 5′-GGTAATACGACTCACTATAGG_GTGTGTTTGAAGCTGCTCGA-GTTGATGTGGGTCGGCTTA-3′; p75/NGFR forward 5′-TCGATGAAGAGCCATGTTTG-3′, p75/NGFR T7 reverse 5′-GGTAATACGACTCACTATAGG_GCCTCATCTGGGAGTGGTAA-3′) and a DIG RNA Labeling Mix, 10× concentration (Roche, Basel, Switzerland) containing digoxigenin labeled uracil. After the IVT reaction, the product was briefly centrifuged and incubated at 37 °C for 1 h. Then 1 µL of turbo DNase 1 was added, the sample was mixed well and incubated for 15 min at 37 °C. One microliter of EDTA 0.5 M was added to stop the reaction. Reaction product was analyzed by gel electrophoresis and quantified.
de Girolamo P., Leggieri A., Palladino A., Lucini C., Attanasio C, & D’Angelo L. (2020). Cholinergic System and NGF Receptors: Insights from the Brain of the Short-Lived Fish Nothobranchius furzeri. Brain Sciences, 10(6), 394.
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2

Zebrafish ache Gene Expression Analysis

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Zebrafish ache coding sequence specific PCR fragment was amplified and T-A cloned into pGEMT-easy vector (Promega, Madison, USA). Plasmids were linearized by restriction enzyme digestion, purified and digoxygenin-labeled anti-sense RNA was produced using MAXIscript SP6/T7 in vitro transcription kit (Invitrogen, USA). Eluted probe was purified using LiCl method. ache whole-mount in situ hybridization (WISH) was performed using standard protocols64 (link).
Avci M.E., Keskus A.G., Targen S., Isilak M.E., Ozturk M., Atalay R.C., Adams M.M, & Konu O. (2018). Development of a novel zebrafish xenograft model in ache mutants using liver cancer cell lines. Scientific Reports, 8, 1570.
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3

In situ Hybridization of wdr81 in Fish Brain and Eye

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The distribution of wdr81 expression in adult brain and eye was determined using in situ hybridization. Ten micrometer thick coronal sections were taken from the eye and brain of a 10 month old male fish using a cryostat (CM 1850, Leica, Germany). The in situ hybridization experiment was carried out as described previously [1 (link)]. The antisense probe was synthesized as mentioned previously in whole mount in situ hybridization section. The same plasmid construct was used as a template for synthesis of the sense probe. NcoI (FD0573, Thermo Fisher Scientific) and ApaI (ER1411, Thermo Fisher Scientific) were utilized for linearization of the plasmid construct and the sense RNA probe was made using the SP6 enzyme mix (AM1320, MaxiScript SP6/T7 In Vitro Transcription Kit, Ambion, USA) and DIG-labelling mix (11277073910, DIG RNA Labelling Mix, Roche, Germany). The slides were visualized with the brightfield upright microscope (Fluorescent and DIC equipped upright microscope, Zeiss, Germany).
Doldur-Balli F., Ozel M.N., Gulsuner S., Tekinay A.B., Ozcelik T., Konu O, & Adams M.M. (2015). Characterization of a novel zebrafish (Danio rerio) gene, wdr81, associated with cerebellar ataxia, mental retardation and dysequilibrium syndrome (CAMRQ). BMC Neuroscience, 16, 96.
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4

Spatio-temporal Alx3 Expression Mapping

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Mouse Alx3 fragment plasmid (1,856-kb-cDNA cloned from E16 cDNA library), a generous gift from Dr. Frits Meijlink, was used to map Alx3 from E12.5 to P7. Following transformation and linearization with EcoRI or HindIII (New England BioLabs, Ipswich, MA, USA), antisense and sense RNA probes were synthesized by transcripting with Sp6 and T7 polymerases (MAXIscript® Sp6/T7 In Vitro Transcription Kit, Ambion, Austin, TX, USA), following manufacturer instructions using Digoxigenin (DIG)-11-UTP (Roche, Mannheim, Germany). The mandibular incisor and first molar tooth organs at E12.5, E14.5, E15.5, E16.5, E18.5, P1, P3, and P7 were harvested by gestation timing of CD-1 pregnant mice (Charles River, Newark, NJ, USA). The isolated tooth germs were dissected under surgical microscope in cold DEPC-treated PBS (pH=7.4), and processed for fixation with 4% paraformaldehyde for 24h at 4°C with gentle rotation. The isolated tooth germs were then exposed to 30% w/v sucrose and incubated at 4°C prior to mounting. Postnatal tooth organs were first decalcified with 0.5M EDTA (pH=8.0) and dehydrated with 30% w/v sucrose. Frozen samples at 8-pm thickness were sectioned for in situ hybridization. NBT/BCIP from DIG Nucleic Acid Detection Kit (Roche) was used to detect Alx3 expression. Alx3 transcripts were treated at 65°C with 1:5000 diluted anti-DIG-AP antibody (Roche).
He L., Zhou J., Chen M., Lin C.S., Kim S.G., Zhou Y., Xiang L., Xie M., Bai H., Yao H., Shi C., Coelho P.G., Bromage T.G., Hu B., Tovar N., Witek L., Wu J., Chen K., Gu W., Zheng J., Sheu T.J., Zhong J., Wen J., Niu Y., Cheng B., Gong Q., Owens D.M., Stanislauskas M., Pei J., Chotkowski G., Wang S., Yang G., Zegarelli D.J., Shi X., Finkel M., Zhang W., Li J., Cheng J., Tarnow D.P., Zhou X., Wang Z., Jiang X., Romanov A., Rowe D.W., Wang S., Ye L., Ling J, & Mao J.J. (2019). Parenchymal and Stromal Tissue Regeneration of Tooth Organ by Pivotal Signals Reinstated in Decellularized Matrix. Nature materials, 18(6), 627-637.
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