RNA substrates were in vitro transcribed using the MAXIscript® SP6 Kit (Ambion) to generate 248- and 277-nt transcripts mapping to the 3′ ends of MTATP6 and MTND3, respectively. Polyadenylation reactions, product separation and visualization were performed as described for high-resolution assays.
Maxiscript sp6 kit
Manufactured by Thermo Fisher Scientific
The MAXIscript® SP6 Kit is a laboratory product designed for the in vitro transcription of RNA from SP6 promoter-driven DNA templates. It provides the necessary components, including the SP6 RNA polymerase enzyme, to generate RNA transcripts from linear or circular DNA templates.
Automatically generated - may contain errors
3 protocols using maxiscript sp6 kit
1
In vitro Transcription of mtRNA Fragments
2
Ribonuclease Protection Assay Probes
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The α-globin probe is a 174-bp fragment encompassing the 3′ part of exon 3 inserted into the polylinker region of pTRI-amp-18 (Ambion) and was generated by in vitro transcription, using a Maxiscript T7 or SP6 kit (Ambion), according to the manufacturer's standard protocol. The β-globin probe was produced using a Maxiscript SP6 kit (Ambion) and consists of a 170 bp fragment encompassing the last 20 bp of intron 2, the entire exon 3 coding region, and the first 21 bp of 3′ UTR, which was amplified by PCR and inserted into the polylinker region of pGEM3 (Promega). The puroR probe was generated using a Maxiscript T7 kit (Ambion), comprises a 280 bp puromycin-resistance gene fragment cloned into pGEM3 and protects 197 nt of the puromycin-resistance mRNA. Samples were processed as described elsewhere (41 (link),44 (link)).
3
CRISPR/Cas9 mRNA and sgRNA Synthesis
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The zCas9 expression plasmid, pGH-SP6-zCas9 [1 (link)], was enzymatically linearized using XbaI and subsequently utilized for in vitro synthesis of Cas9 mRNA employing the mMACHINE SP6 Ultra kit (Ambion). The resulting zCas9 mRNA was purified utilizing the same kit. According to previously described criteria [2 (link)], the single guide RNA (sgRNA) was designed. To minimize potential off-target effects, the CRISPR/Cas9 design tool (http://chopchop.cbu.uib.no/ ) was employed to identify specific target sequences. The sequences of the designed sgRNA is 5′-TCACACTTTTACCCCGGGGT TGG-3’. The sgRNA was annealed and subsequently cloned into the PSP6-sgRNA vector downstream of the SP6 promoter. To synthesize the sgRNA, the MAXIscript SP6 Kit (Ambion) was utilized, and purification of the sgRNA was performed using the mirVana™ miRNA Isolation Kit (Ambion).
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