In-cell NMR sample was prepared by direct microinjection 50 nl aliquot of the stock solution (3 and 5 mM of 12-mer RNA) into the oocyte cell (∼150 and ∼250 μM intracellular concentration). Approximately 150 Xenopus laevis oocytes were injected by using an IM-30 Electric Microinjector (NARISHIGE, Tokyo). After injection, the oocytes were transferred to a disposable dish, washed carefully with oocyte stocking buffer (15 mM Tris-HCl (pH7.6), 88 mM NaCl, 1 mM KCl, 0.4 mM CaCl2, 0.3 mM Ca(NO3)2, 0.8 mM MgCl2 and 2.4 mM NaHCO3). The injected oocytes were transferred to a Shigemi tube (Shigemi 5 mm Symmetrical NMR microtube) and kept in an oocyte stocking buffer containing 10% of D2O. Sample for measurement in lysate was prepared as follows: after the in-cell NMR measurements, oocytes were mechanically crushed and used for NMR measurement.
Im 30 electric microinjector
Manufactured by Narishige
Sourced in Japan
The IM-30 Electric Microinjector is a laboratory device designed for the precise injection of small volumes of liquid into samples or cells. It provides controlled injection of reagents, solutions, or other materials in a range of research applications.
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Lab products found in correlation
2 protocols using im 30 electric microinjector
1
In-cell NMR of 12-mer RNA in Xenopus Oocytes
2
Knockdown of Ribosomal Protein Genes
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Two types of MOs were obtained from Gene Tools, LLC (OR, USA), to knock down the rpl10a gene. The AUG MO (MOaug: 5′-GACCTTGCTCATTTTGGCGTGATAT-3′) contained 13 bp of the 5′ UTR, the translation start site, 2 bp of exon 1 and 7 bp of exon 2 to block Rpl10a protein translation. Splice MOs were designed to target pre-mRNA splicing at exon 5/intron 5 (MOsp: 5′-ATCACAAATATAGACATACCTTCTT-3′). The rps19 MOaug (5′-CACTGTTACACCACCTGGCATCTTG-3′) was used to develop an anemia phenotype described previously24 (link). MOs (MOaug at 0.5 µg/µl; MOsp at 5 µg/µl) were injected into one- or two-cell-stage embryos (wild-type AB line) using an IM-30 Electric Micro-injector (Narishige, Japan). The morphology was observed at 25 and 50 hpf.
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