RNAs for GCaMP6s and PKCδ-GFP were synthesised from pCS2-based plasmids containing the respective coding sequences (Sivak et al., 2005 (link); Chen et al., 2017 (link)). These were linearised with NotI (NEB), and RNA in vitro transcribed with mMESSAGE mMACHINE SP6 Transcription Kit (Ambion). RNA for Tol2 was generated from the pT3Ts-Tol2 plasmid, linearised with SmaI (NEB), and transcribed with the mMESSAGE mMACHINE T3 Transcription Kit (Ambion). RNA for injection was purified by lithium chloride precipitation.
Mmessage mmachine t3 transcription kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MMESSAGE mMACHINE T3 Transcription Kit is a reagent kit designed to facilitate in vitro transcription of RNA from DNA templates. The kit contains the necessary components, including the T3 RNA polymerase enzyme, ribonucleotides, and other reagents, to efficiently produce high-quality RNA.
Automatically generated - may contain errors
Lab products found in correlation
Poly a tailing kit, by Thermo Fisher Scientific (6 mentions)
Rneasy mini kit, by Qiagen (6 mentions)
Pt3ts ncas9n plasmid, by Addgene (5 mentions)
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (5 mentions)
Pt7 grna, by Addgene (4 mentions)
Pt3ts ncas9n, by Addgene (4 mentions)
Xbai, by New England Biolabs (4 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (4 mentions)
Roboinject system, by MultiSciences Biotech (4 mentions)
Mmessage mmachine sp6 transcription kit, by Thermo Fisher Scientific (2 mentions)
Maxiscript t7 kit, by Thermo Fisher Scientific (2 mentions)
Megaclear transcription clean up kit, by Thermo Fisher Scientific (2 mentions)
Megashortscript t7 transcription kit, by Thermo Fisher Scientific (2 mentions)
Megaclear kit, by Thermo Fisher Scientific (2 mentions)
Neb monarch rna clean up kit, by New England Biolabs (2 mentions)
Mmessage mmachine t7 kit, by Thermo Fisher Scientific (1 mentions)
T7 rna polymerase, by Promega (1 mentions)
Szx16 stereomicroscope, by Olympus (1 mentions)
Microinjection setup, by Narishige (1 mentions)
Xbai, by Promega (1 mentions)
51 protocols using mmessage mmachine t3 transcription kit
1
RNA Synthesis for GCaMP6s, PKCδ-GFP, and Tol2
2
TALEN and CRISPR-Cas9 mRNA Synthesis
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In vitro transcription of TALEN mRNAs was performed using the mMessage mMachine T7 kit (Ambion) using pJDS-TALEN constructs linearized with PmeI and phenol-chloroform purified, and were polyadenylated using the Poly(A) Tailing Kit (Ambion), according to the manufacturer’s instructions. In vitro transcription of Streptococcus pyogenes Cas9 mRNA was performed using the mMessage mMachine T3 transcription kit (Ambion), using the pCS2-nCas9n expression plasmid from Addgene (Jao et al. 2013 (link)) linearized with XbaI and purified with phenol-chloroform. The resulting capped PolyA mRNAs were purified by acid-phenol-chloroform extraction and resuspended in RNase-free water following isopropanol precipitation. sgRNAs were in vitro transcribed from sgRNA-containing DR274 vectors linearized with DraI and purified by phenol-chloroform extraction, using T7 RNA polymerase (Promega) according to the manufacturer’s instructions. RNAs were quantitated by spectrophotometry (NanoDrop 1000) and diluted in RNase-free water to a final concentration of 0.5 μg/μl for cry2 TALENs and clk TALENs, 0.5 μg/μl for Cas9, and of 0.1 μg/μl for cry2 and clk sgRNAs.
3
Recombinant Hsp104 Protein Injection in C. elegans
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Two different HSP104 mRNAs were used in this study. One was derived from the coding region of pRS416GAL-HSP104A503V (Jackrel and Shorter, 2014 (link)), which was subcloned into pRN3P (a gift from John Gurdon; Zernicka-Goetz et al., 1996 (link)) where the original GFP was substituted for mRed to make C-terminal RFP fusion, and contains 5′ and 3′ untranslated regions from ß-globin. The mutant Hsp104 was derived from pRS313- HSP104K218;K620T (Patino et al. 1996 (link); Addgene), which was cloned into the modified pRN3P vector. Transcription templates were generated using PCR, and following purification (Qiagen), mRNA was generated from the PCR product using mMESSAGE mMACHINE T3 Transcription Kit (AM1348, Ambion), precipitated with LiCl, and diluted to 0.1 µg/µl in DEPC water for injection into adult C. elegans hermaphrodite germlines. Recombinant Hsp104 protein (LifeSpan BioSciences) was injected following dilution to 0.1 mg/ml in a buffer containing 20 mM Tris HCl, 100 mM NaCl, and 2 mM EDTA, pH 8.
4
CRISPR-Cas9 Knockout of Zebrafish Opsins
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Guide RNAs (SI Appendix, Fig. S4 ) were generated as described previously (28 (link)). The plasmids pT7-gRNA and pT3TS-nCas9n were obtained from Addgene (nos. 46759 and 46757). The oligonucleotide sets designed for guide RNAs were as follows: 5′-TAGGGTGGCGGTGTCCTGGGTC-3′ and 5′-AAACGACCCAGGACACCGCCAC-3′ for zebrafish parapinopsin guide RNA and 5′-GGCGTATGAGCGCTATAACG-3′ and 5′-AAACCGTTATAGCGCTCATACG-3′ for zebrafish parietopsin guide RNA. Annealed oligonucleotide sets were inserted into pT7-gRNA, which had been linearized by BsmB I. Guide RNAs were transcribed by using the MEGAscript T7 Transcription Kit. Cas9 mRNA was transcribed in vitro by using the mMESSAGE mMACHINE T3 Transcription Kit (Ambion). Guide RNA (∼50 pg) and Cas9 mRNA (∼150 pg) were coinjected into embryos during the one-cell stage. Mutations in the zebrafish parapinopsin and parietopsin nucleotide sequences were confirmed by a heteroduplex mobility assay (29 (link)). Stop codons appear after Val224 and Ser135 in the PP−/− and PT−/− mutants, respectively. Opsin(s)-deficient fish were obtained by mating the established KO strains.
5
Tol2 Transgenesis in Zebrafish
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Wild‐type strain AB zebrafish embryos were injected at the one‐cell stage into the cell cytoplasm with 25pg tol2 mRNA and 25 pg circular plasmid in 1 nL. Tol2 mRNA was synthesised from pT3‐Tol2 linearised with SmaI (NEB) with mMESSAGE mMACHINE T3 Transcription Kit (Ambion) and purified with LiCl extraction. Injected embryos with the strongest expression of mosaic GFP were grown into adults and out‐crossed to screen for germline transmission.
6
Microinjection of Fluorescent Markers in Oocytes
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Microinjection needles (World Precision Instruments, 1B100-4) were pulled with a P-97 needle puller (Sutter Precision Instruments) and mounted on a microinjection setup (Narishige) attached to an Olympus SZX16 stereomicroscope. Dechorionated oocytes were placed in agarose wells. In vitro mRNA transcription was performed using the mMESSAGE mMACHINE T3 Transcription Kit (Ambion, AM1343). The following mRNAs were injected in dechorionated unfertilized oocytes: Utrophin-Venus (a gift from Alex McDougall, Sorbonne University), Utrophin-mScarlet (this study), iMyo-YFP (a gift from Edwin Munro, University of Chicago), iMyo-mScarlet (this study), iMyo-mKO2 (this study), Utrophin-mNeonGreen (this study) and CaMypt51 (link). mRNAs were injected at a concentration of 1–1.5 µg ml−1 the evening before the experiments, except for CaMypt mRNA, which was injected 3–4 h before the experiment.
7
CRISPR-Cas9 mRNA and sgRNA Synthesis
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Template DNA for short guide RNAs (sgRNAs) synthesis was digested with DraI, and sgRNAs were transcribed using the MAXIscript T7 kit (Life Technologies). The pT3TS-nCas9n plasmid (Addgene) (Jao et al., 2013 (link)) was linearized with XbaI (Promega) and mRNA synthesized with the mMessage mMachine T3 Transcription Kit (Ambion). Transcription reactions were incubated in a water bath at 37°C for 2 h or longer. To digest template DNA, 1 μL of TURBO DNase was added and reaction incubated for a further 15 min at 37°C. The Cas9 mRNA was polyadenylated using the polyA tailing kit (Ambion). Cas9 mRNA and sgRNAs were purified using either the RNeasy Minikit (Qiagen) or Zymo columns.
8
Microinjection of CRISPR-Cas9 Components for Zebrafish Genome Editing
9
CRISPR Cas9 sgRNA Synthesis Protocol
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sgRNAs for each target were generated by the oligonucleotide assembly method as described earlier (13 (link)). RNAs were synthesized using the HiScribe™ T7 Quick High Yield RNA Synthesis Kit (New England Biolabs) with an incubation time of 8 h for the in vitro transcription reaction. RNAs were purified with the RNA Clean & Concentrator™-5 kit (Zymo Research) and eluted into 20 μl water. Cas9 mRNA was synthesized from the pT3TS-nCas9n plasmid (Addgene plasmid # 46757) (12 (link)) following XbaI digestion using the mMessage mMachine T3 Transcription Kit (Ambion) and recovered by lithium chloride precipitation.
10
In vitro Synthesis of gRNAs and Cas9 mRNA
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For the in vitro synthesis of gRNAs, the T7 promoter was inserted upstream of gRNAs by PCR using oligomers as previously described31 (link), and 5′-GG transcription start region was arbitrarily created (Supplementary Table S4 ). The T7-gRNAs were gel-purified for in vitro transcription. Both std-gRNAs and tru-gRNAs were transcribed in vitro with the MEGAshortscript Kit (AM1354, Ambion). The pCAG-T3-hCAS-pA (#48625, Addgene) plasmid was digested by NruI, and the DNA fragment encoding Cas9 was recovered by gel extraction for in vitro transcription. Cas9 mRNA was transcribed with the mMESSAGE mMACHINE® T3 Transcription Kit (AM1348, Ambion). All RNAs were purified with the MEGAclear Transcription Clean-Up Kit (AM1908, Ambion) and stored at −80° C until use.
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