The complementary DNA (cDNA) encoding mouse ASIC1a (mASIC1a) was used as previously described (Lynagh et al., 2017 (link)). Site-directed mutagenesis was performed using PfuUltraII Fusion polymerase (Agilent) and custom DNA mutagenesis primers (Eurofins Genomics). All sequences were confirmed by sequencing of the full coding frame (Eurofins Genomics or Macrogen). cDNAs were linearized with EcoRI, and capped cRNA was transcribed with the Ambion mMESSAGE mMACHINE SP6 kit (Thermo Fisher Scientific).
Ambion mmessage mmachine sp6 kit
Manufactured by Thermo Fisher Scientific
The Ambion mMESSAGE mMACHINE SP6 kit is a laboratory tool used for the in vitro transcription of messenger RNA (mRNA) from DNA templates. The kit includes reagents and protocols for the efficient synthesis of capped and polyadenylated mRNA, which can be used for various downstream applications such as gene expression studies, RNA interference, and more.
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4 protocols using ambion mmessage mmachine sp6 kit
1
Generation of Mutant mASIC1a Constructs
2
Construct RNA Injection Protocol
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Constructs were cloned into pCSDest (Villefranc et al., 2007 (link)), linearized, transcribed using the Ambion mMessage mMachine SP6 Kit (Thermo Fisher Scientific), and purified with the RNeasy MinElute RNA Cleanup Kit (Qiagen). RNA was quantified based on absorbance at 260 nm and injected with 0.1% phenol red into 1–2 cell embryos in a volume of 1–2 nl.
3
Mutagenesis of Ion Channel cDNAs
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Mouse ASIC1a cDNA in the pSP64 vector was obtained from Dr. Marcelo Carattino (University of Pittsburgh). The rat ASIC3 clone in the pRSSP6009 vector was provided by Dr. Stefan Gründer ( RWTH Aachen University). Mutations were introduced via site-directed mutagenesis using PfuUltraII Fusion polymerase (Agilent) and custom-made DNA mutagenesis primers (Eurofins Genomics). Mutagenesis was confirmed by sequencing the full coding frame (Eurofins Genomics or Macrogen). All cDNAs were linearized using EcoRI. For transcription to capped cRNA, the Ambion mMESSAGE mMACHINE SP6 kit (Thermo Fisher Scientific) was used.
4
Mutagenesis and Transcription of Mouse ASIC1a
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Molecular biology.
The complementary DNA (cDNA) encoding mouse ASIC1a (mASIC1a) was used as previously described (Lynagh, Flood et al. 2017) . Site-directed mutagenesis was performed using PfuUltraII Fusion polymerase (Agilent) and custom DNA mutagenesis primers (Eurofins Genomics). All sequences were confirmed by sequencing of the full coding frame (Eurofins Genomics or Macrogen). cDNAs were linearized with EcoRI and capped cRNA was transcribed with the Ambion mMESSAGE mMACHINE SP6 kit (Thermo Fisher Scientific).
The complementary DNA (cDNA) encoding mouse ASIC1a (mASIC1a) was used as previously described (Lynagh, Flood et al. 2017) . Site-directed mutagenesis was performed using PfuUltraII Fusion polymerase (Agilent) and custom DNA mutagenesis primers (Eurofins Genomics). All sequences were confirmed by sequencing of the full coding frame (Eurofins Genomics or Macrogen). cDNAs were linearized with EcoRI and capped cRNA was transcribed with the Ambion mMESSAGE mMACHINE SP6 kit (Thermo Fisher Scientific).
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