Expressing Slo2.1, Kir6.2, and SUR1 in Oocytes

cDNA for human Slo2.1 (KCNT2, NCBI Genbank accession no. NM_198503), kindly provided by L. Kaczmarek (Yale University), was subcloned into the psGEM oocyte expression vector as described (Dai et al. 2010 (link)). K1031A Slo2.1 was generated by using the QuikChange site‐directed mutagenesis kit (Agilent Technologies) and confirmed by DNA sequencing by the University of Utah sequencing core facility. Human Kir6.2 (KCNJ11) and rat SUR1 (ABCC8) cDNAs in the pBF vector were kindly provided by F. Ashcroft (University of Oxford). Plasmids were linearized with SfiI (Slo2.1) or Mlu1 (Kir6.2, SUR1). cRNAs were prepared using the mMessage mMachine T7 kit (Slo2.1) or SP6 kit (Kir6.2, SUR1) (Ambion, Life Technologies, Grand Island, NY). Concentrations of cRNA were measured with the RiboGreen RNA quantitation kit (Invitrogen, Life Technologies).

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Garg P, & Sanguinetti M.C. (2014). Intracellular ATP does not inhibit Slo2.1 K+ channels. Physiological Reports, 2(9), e12118.

Publication 2014

QuikChange site‐directed mutagenesis kit mMessage mMachine T7 kit RiboGreen RNA quantitation kit

Cdnas Crna Human Oocyte Plasmids Site directed mutagenesis Vector

Corresponding Organization : University of Utah

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Variable analysis

independent variables

Generation of K1031A Slo2.1 mutant using site-directed mutagenesis

dependent variables

Expression of Slo2.1, Kir6.2, and SUR1 proteins in oocytes

control variables

Wildtype Slo2.1 cDNA used as control
Kir6.2 and SUR1 cDNAs used as additional experimental components

controls

Wildtype Slo2.1 cDNA used as positive control
No additional negative controls mentioned

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