Abi prism 3130xl genetic analyzer

Table 2 contains a list of the expression vectors, restriction enzymes and promoters used in this study. The plasmids encoding for hAQP1, hAQP1_FLAG, hAQP1_C189S mutant, rAQP3, hAQP7, hAQP8 or rAQP9 were transformed into TOP10 competent cells. All of the plasmids were sequenced using the BigDye kit and the ABI Prism 3130XL Genetic Analyzer (Hitachi, Tokyo, Japan). Plasmid DNA was purified using either miniprep or midiprep kits (Qiagen). DNA concentration and purity were determined spectrophotometrically on a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific).
Plasmids were linearized using the respective restriction enzyme (Table 2) and restriction-digested products were purified using the QIAquick PCR purification kit (Qiagen). The capped RNAs (cRNAs) were transcribed using either a T3 or T7 mMessage mMachine kit (Ambion, Austin, USA) depending on the promoter present on the linearized DNA (Table 2). The cRNA was then purified and concentrated using the RNeasy MinElute RNA Cleanup kit (Qiagen) and quantified by measuring the absorbance at 260 nm using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific).

The UT-A3 (AF258602), UT-A2 (AF367359) and UT-B (AF448798) were a gift from Dr Bryce MacIver, Harvard Medical School, MA, USA. DNA sequences encoding C-terminally c-Myc tagged murine UTs: mUT-B, mUT-A2 and mUT-A3 were subcloned into the P7TS expression vector. The resulting plasmids were transformed into TOP10 competent cells via heat shock, and purified with a DNA Miniprep kit (part #28104, Qiagen, Valencia, CA, USA). All of the plasmids were sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit (part #4337455, Applied Biosystems, Foster City, CA, USA) and an ABI Prism 3130XL Genetic Analyzer (HITACHI, Tokyo, Japan).
All UT-encoding cDNAs were linearised with XbaI restriction enzyme (part #R0145S, New England Biolabs, Ipswich, MA, USA) and purified using the QIAquick PCR Purification Kit (part #27106, Qiagen). The linearised and purified DNAs were transcribed into capped RNA (cRNA) using the T7 mMachine Kit (part #AM1344, Ambion, Austin, TX, USA) and the cRNAs were purified with the RNeasy MinElute RNA Cleanup Kit (part #74204, Qiagen). The concentration and purity of all DNAs and RNAs were quantified using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).