Dna gel loading dye 6

Agarose gel was made using 3 g agarose and 120 mL TBE buffer (2.5% gel). Ingredients were mixed and microwaved for 2.5 min. Six microliters of Roti^®-GelStain (Carl Roth, Karlsruhe, Germany, ref. no. 3865.1) was added and the gel was poured into a gel chamber. Twenty microliters of each cDNA sample was mixed with 4 µL DNA Gel Loading Dye 6× (ThermoFisher, Waltham, MA, USA, ref. no. R0611) and briefly centrifuged at 2000 rpm. Samples and GeneRuler 100 bp DNA Ladder (ThermoFisher, Waltham, MA, USA, ref. no. SM0241) were added to the agarose gel and electrophoresis was started. Gel Doc 2000 (BIO-RAD, Hercules, CA, USA) was used for imaging.

For gel electrophoretic analysis, 1 µl DNA Gel Loading Dye (6 ×) (Thermo Fisher Scientific; SM0334) was added to 5 µl of the purified samples. For band visualization, a 2% agarose gel was prepared in 1 × TAE buffer (50 × TAE buffer; Jena Bioscience; BU-119–50) containing 1 µl SYBR™ Safe DNA Gel Stain (Thermo Fisher Scientific; S33102) per 50 ml gel to visualize the band. A total volume of 6 µl of the purified and prepared samples were used for electrophoresis which ran at 95 V for 35 min. Detection was done via E-Box gel imaging system (Vilber). GeneRuler DNA Ladder Mixture, Ready to Use (Thermo Fisher Scientific; SM0334) was used for calculation of the fragment length.

The purity of the collected renal tubules and the expression of NBCe2 was determined by RT-PCR (see Supplementary Table S1 for primer sequences and product sizes). RNA from the tubules was purified using an RNeasy micro kit (Qiagen, Copenhagen, Denmark) according to the manufacturer’s instructions. The concentration of purified RNA was determined by absorbance at 260 nm using a NanoDrop ND-2000 (Fisher Scientific) and 20–40 ng of RNA was reverse transcribed by iScript Reverse Transcription Supermix (Bio Rad, Copenhagens, Denmark). PCR amplification was performed for each transcript by mixing cDNA with 1 pmol of primers and 5× HOT FIREPol Blend Master Mix (Solis BioDyne). The PCR reaction was performed for 35 cycles after 15 min at 95°C: denaturation was performed for 30 s at 95°C, annealing at 60°C for 30 s, and elongation at 72°C for 1 min. PCR products were mixed with 2 μl of DNA Gel Loading Dye (6×; Thermo Scientific) containing 0.05% 10000 × GelRed (Biotium, BioNordika Denmark, Herlev, Denmark) nucleic acid gel stain, separated by 1% agarose gel electrophoresis, and photographed under ultraviolet illumination.