Methods for site-directed mutagenesis have been previously reported24 (link),30 (link). The hERG1 constructs were transfected into mammalian HEK cells. Single- and double-mutant constructs of hERG1 were produced using conventional overlap PCR with primers synthesized by Sigma Genosys (Oakville, Ontario, Canada) and sequenced using Eurofins MWG Operon (Huntsville, AL) (Supplementary Table 1 ). Constructs were linearized with XbaI restriction endonuclease and cRNA was transcribed in vitro using the mMessage mMachine T7 Ultra cRNA transcription kit (Ambion, Austin, TX).
Mmessage mmachine t7 ultra crna transcription kit
Manufactured by Thermo Fisher Scientific
The MMessage mMachine T7 Ultra cRNA transcription kit is a laboratory equipment product designed for the in vitro synthesis of capped and polyadenylated cRNA from linearized DNA templates. The kit contains the necessary components, including the T7 RNA polymerase, ribonucleotides, and other buffers, to facilitate the transcription process.
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Lab products found in correlation
5 protocols using mmessage mmachine t7 ultra crna transcription kit
1
Site-Directed Mutagenesis of hERG1 Constructs
2
Generating hERG1 Mutant Constructs
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The site-directed mutagenesis methods utilized in this work have been previously reported (Lees-Miller et al., 2015 (link); Wang et al., 2016 (link)). The hERG1 constructs were transfected into mammalian HEK cells. Conventional overlap PCR with primers were synthesized by Sigma Genosys (Oakville, Ontario, Canada) and sequenced by using Eurofins MWG Operon (Huntsville, AL) to create the single- and double-point mutant constructs of hERG1. Following this, XbaI restriction endonuclease was used to linearize and the cRNA was transcribed in vitro using the mMessage mMachine T7 Ultra cRNA transcription kit (Ambion, Austin, TX).
3
Xenopus Oocyte Expression of hERG1a WT and R56Q
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For expression in Xenopus laevis oocytes, hERG1a WT or R56Q mutant channel cDNA, subcloned into a pBluescript SKII expression vector, was linearized using XbaI to synthesize complementary RNA (cRNA) using the mMessage mMachine T7 Ultra cRNA Transcription Kit (Ambion). Mutations were generated by conventional overlap extension PCR using mutagenic primers synthesized by Sigma-Genosys and confirmed by sequencing (Eurofins MWG Operon). For expression in human embryonic kidney (HEK) cells, hERG1a WT or R56Q mutant channel cDNA was subcloned into a pcDNA3 vector.
4
Cloning and Mutagenesis of hERG1a Channel
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hERG1a channel cDNA was subcloned into a pBluescript SKII expression vector. The D509A mutation was generated by conventional overlap extension PCR. Mutagenic primers were synthesized by Sigma-Genosys. The mutation was confirmed by sequencing using Eurofins MWG Operon. The XbaI restriction enzyme was used to linearize the construct for subsequent synthesis of cRNA with the mMessage mMachine T7 Ultra cRNA Transcription Kit (Ambion).
5
Constructing hERG Ion Channel Mutants
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All hERG channel constructs were subcloned into the expression vector pBluescript SKII and expressed in Xenopus laevis oocytes. Conventional overlap extension PCR with primers synthesized by Sigma Genosys (Oakville, ON) were used for constructing hERG I663P and I663P/S620T mutant channels. The mutant channels were sequenced using Eurofins MWG Operon (Huntsville, AL) to ensure no errors were integrated during PCR cycling. Once the desired mutation was achieved, constructs were linearized using XbaI restriction endonuclease to create a template for in vitro transcription of cRNA. cRNA was transcribed from linear cDNA using the mMessagemMachine T7 Ultra cRNA transcription kit (Ambion, Austin, TX).
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