The Y170A mutant of cyclin B1 was made using the GeneEditor (Promega) in vitro mutagenesis system according to the manufacturer’s protocol. Microtubules were visualised by expression of mRNA coding for the microtubule binding protein Map7 fused to GFP (Prodon et al., 2010 (link)). Wild type human cyclin B1 (NM_031966), human securin (AF095287.1) and human CDK1 K33A (a gift from Jonathon Pines) sequences were amplified by PCR as previously described (Madgwick et al., 2004 (link)). Further cyclin B1 mutations were generated by primer overhang extension PCR. Δ90 cyclin B1 was cloned into a pRN3 vector (a gift from Patrick Lamaire; (Lemaire et al., 1995 (link)) while all other amplified sequences were cloned into a modified pRN3 vector designed to produce mRNA transcripts C-terminally coupled to Venus or Cerulean fluorescent proteins (Levasseur, 2013 ). The CDK1 FRET sensor (Addgene plasmid #26064) and inactive sensor (a gift from Jonathon Pines) sequences were amplified by PCR and cloned into the pRN3 vector. Maximal stability was conferred on all cRNA constructs by the presence of a 5′ globin UTR upstream and both 3′UTR and poly (A)–encoding tracts downstream of the gene. cRNA for microinjection was synthesized using T3 mMESSAGE mMACHINE (Ambion) and dissolved in nuclease-free water to the required micropipette concentration.
T3 mmessage mmachine
Manufactured by Thermo Fisher Scientific
The T3 mMESSAGE mMACHINE is a lab equipment product designed for in vitro transcription. It is used to synthesize capped and polyadenylated mRNA from DNA templates.
Automatically generated - may contain errors
Lab products found in correlation
Xbai, by New England Biolabs (2 mentions)
Poly a tailing kit, by Thermo Fisher Scientific (2 mentions)
Phenol red, by Merck Group (2 mentions)
Geneeditor, by Promega (1 mentions)
Beckman liquid scintillation counter, by Beckman Coulter (1 mentions)
Micromanipulator, by Narishige (1 mentions)
Femtojet microinjector, by Eppendorf (1 mentions)
Dmirb inverted microscope, by Leica (1 mentions)
Pcr purification kit, by Thermo Fisher Scientific (1 mentions)
Megascript t7, by Thermo Fisher Scientific (1 mentions)
Golden gate kit, by Addgene (1 mentions)
Poly a polymerase tailing kit, by Illumina (1 mentions)
Rneasy cleanup kit, by Qiagen (1 mentions)
Rneasy micro purification kit, by Qiagen (1 mentions)
Cdna synthesis kit, by Takara Bio (1 mentions)
Rnaeasy kit, by Qiagen (1 mentions)
T7 kits, by Thermo Fisher Scientific (1 mentions)
13 protocols using t3 mmessage mmachine
1
Cyclin B1 Mutagenesis and Visualization
2
Heterologous Expression of hENTs in Xenopus Oocytes
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The mutant plasmids were linearized by either the NotI or SacI restriction enzyme. The digested products were purified by phenol-chloroform extraction. The cRNAs were synthesized using a T3 mMESSAGE mMACHINE (Ambion, Foster City, CA) transcription kit following the manufacturer’s instructions. The cRNAs were purified by lithium precipitation. Fifty nanoliters (400–800 ng/µl) of equal amounts of cRNAs of various hENTs were injected into oocytes 24 hours after defolliculation. The injected oocytes were incubated at 15°C for 24 hours before transport assays were performed. Uptake of radiolabeled substrates was measured after 30 minutes of incubation in transport buffer (100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES, pH 7.4) at room temperature. Uptake was terminated by washing the oocytes three times with arrest buffer (100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 20 mM uridine, pH 7.4). Individual oocytes were shaken overnight in 10% SDS for complete dissolution, and the total radioactivity was then quantified by a Beckman liquid scintillation counter (Beckman Coulter, Brea, CA).
3
Microinjection of PLK1 and CIP2A Mutants
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CIP2A-S904A and CIP2A-S904D were subcloned into pRN3-mCherry vectors, as described previously (26 (link)). PLK1-T210D clones were obtained from Addgene (#68133) and were subcloned into the pRN3-mCherry vector. PLK1 mRNAs were transcribed in vitro using a T3 mMESSAGE mMACHINE (Ambion) according to the manufacturer's protocol. After purification, mRNAs were diluted to a concentration of approximately 500 ng/μl and microinjected into GV oocytes on the stage of a DMIRB inverted microscope (Leica) using a FemtoJet microinjector (Eppendorf) and micromanipulators (Narishige).
4
Preparation of cRNA for GFP-tagged Proteins
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The cRNA for GFP-tagged securin and GFP-tagged geminin were prepared from the T3 promoter of a pRN3-GFP vector, using a transcription kit (T3 mMESSAGE mMACHINE; Ambion). The cRNA was then polyadenylated and purified in nuclease-free water to a concentration of ∼1 µg/µl before microinjection.
5
Xenopus Oocyte mRNA Injection Protocol
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Batches of Xenopus laevis oocytes were provided by the European Xenopus Resource Centre of the University of Portsmouth (UK). Oocytes were isolated from Xenopus laevis and injected with mRNA using established methods. 34 Briefly, mature adult female frogs were anesthetized with 3-amino benzoic acid ethyl ester (2 g/750 mL) in ice water. Frog ovaries were resected, and their ovarian lobes opened and incubated in OR-2 without calcium (5 mM of HEPES, 82.5 mM of NaCl, 2.5 mM of KCl, 1 mM of MgCl 2 , 1 mM of Na 2 HP0 4 , 100 μg/mL of gentamicin, pH 7.8) with collagenase type IV (2 mg/mL) for 30 minutes at 23°C. Individual oocytes were isolated and transferred to OR-2 containing 1 mM of CaCl 2 and maintained at 18-20°C until injection with mRNA. SGLT1 and GLUT cRNA were prepared by cutting plasmid vectors with appropriate restriction enzymes followed by in vitro transcription utilizing SP6, T7, or T3 mMessage, mMachine (Ambion). cRNA was loaded into capillary pipettes generated using a micropipette puller (P-77, Sutter, Novato, CA), and oocytes were injected using a pressure-controlled injector (Drummond Nanoject 11, USA). Injection volumes were between 30 and 50 nL, and cRNA concentration 0.5-1 mg/mL. Postinjected oocytes were incubated at 20°C in OR-2 containing 1 mM of pyruvate with daily media changes. Experiments were performed on 3-5 days after oocytes were injected with mRNA.
6
Construct pRN3-Emi21−300-mCherry Plasmids
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pRN3-Emi21−300-mCherry plasmids containing the Plk1 recognition site and a degron for a SCF family ubiquitin ligase were constructed from synthetic Emi21–300 gene fragments and subcloned into the pRN3 vector as an mCherry fusion by means of Gibson Assembly50 (link). The plasmids were linearized by digestion with SfiI; then cRNA was synthesized with T3 mMessage mMachine (Life Technologies). In vitro transcripts were purified by phenol-chloroform extraction and isopropyl alcohol precipitation and stored at −80 °C until use.
7
Synthesizing CRISPR-Cas9 mRNA from Addgene Plasmid
8
Cas9-encoding mRNA and sgRNA Synthesis
9
Injecting Plasmid DNA and RNA in Zebrafish
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DNA plasmid and RNA transcript for injection were diluted to final concentration of 30 ng/µl and 200 ng/µl, respectively, with addition of Phenol Red (Sigma-Aldrich) as injection tracer. A bolus of approximately 1/5 of the total cell diameter was injected into each embryo. For DNA injection, the bolus was injected into the cell of embryos at single cell stage (5–25 min-post-fertilisation). For RNA injection, the bolus was injected into the yolk of the embryos up until two-cell stage. The RNA transcript was synthesised by mMESSAGE mMACHINE T3 (Invitrogen) according to manufacturer’s instruction. The RNA transcripts were tagged with poly(A) tail using Poly(A) Tailing Kit (Invitrogen) to extend the stability of mRNA in zebrafish embryos.
10
Transient Gene Expression in Zebrafish
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Transient expression by DNA/RNA microinjection DNA plasmid and RNA transcript for injection were diluted to final concentration of 30ng/µl and 200ng/µl, respectively, with addition of Phenol Red (Sigma-Aldrich) as injection tracer. A bolus of approximately 1/5 of the total cell diameter was injected into each embryo. For DNA injection, the bolus was injected into the cell of embryos at single cell stage (5 to 25 min-post-fertilisation). For RNA injection, the bolus was injected into the yolk of the embryos up until 2-ell stage. The RNA transcript was synthesised by mMESSAGE mMACHINE™ T3 (Invitrogen) according to manufacturer's instruction. The RNA transcripts were tagged with poly(A) tail using Poly(A) Tailing Kit (Invitrogen) to extend the stability of mRNA in zebrafish embryos.
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