Ten micrograms of plasmid DNA was linearized by digestion with Not I–HF. Linearized DNA was purified with the MinElute PCR Purification Kit (Qiagen, catalog no. 28004) according to the manufacturer’s instructions and diluted to 0.5 μg/μl in elution buffer (EB), and 2 μg was used as a template for in vitro transcription. Transcription was performed using the Ribomax T7 Kit (Promega, catalog no. P1320). The reaction was incubated at 37°C for 30 min followed by 15-min DNase (from kit) treatment at 37°C. To reduce the carryover of residual undigested plasmid or DNA fragments, the DNase-treated RNA was transferred to a new tube before purifying RNA using the RNeasy Mini Kit (Qiagen, catalog no. 74014) following the manufacturer’s instructions. The optional on-column DNase digestion step (Qiagen, catalog no. 79254) was included. Capping and polyadenylation were performed using T7 mScript Standard mRNA Production System (CELLSCRIPT, catalog no. C-MSC100625) following the Cap 1 mRNA protocol described in the user’s manual, and the RNA was again purified using the RNeasy Mini Kit without on-column DNase digestion. The expected RNA yield is ~45 to 60 μg per reaction.
Rneasy mini kit
Manufactured by CellScript
The RNeasy Mini Kit is a laboratory equipment designed for the purification of total RNA from various biological samples. It utilizes a silica-membrane-based technology to efficiently capture and purify RNA molecules. The kit provides a simple and reliable method for extracting high-quality RNA suitable for a range of downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using rneasy mini kit
1
In Vitro Transcription of Plasmid DNA
2
Synthesis and Injection of Capped mRNAs
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To synthesize mRNA for microinjection, RNA was transcribed from a linearized plasmid DNA template using the SP6-Scribe Standard RNA IVT Kit (CELLSCRIPT) and purified with the RNeasy Mini Kit (QIAGEN). The m 7 G cap structure was added to the purified RNA using the ScriptCap m 7 G Capping System (CELLSCRIPT). Capped mRNA was purified with the RNeasy Mini Kit. For sfGFP reporter mRNAs, the poly(A) tail was added using the A-Plus Poly(A) Polymerase Tailing Kit (CELLSCRIPT), and the mRNA was then purified with the RNeasy Mini Kit. Purified mRNAs were diluted with water to the following concentrations: sfGFP reporter mRNAs and PACE reporter library 50 ng/µl; MT-Znf598 mRNAs 100 ng/µl; AnsB 50 ng/µl; tandem ORF reporter mRNAs 100 ng/µl. GFP MO was injected as described previously (Mishima and Tomari, 2016) (link). Microinjection was performed using an IM300 Microinjector (NARISHIGE). Approximately 1,000 pl of solution was injected per embryo within 15 min after fertilization.
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