A polymerizable anion receptor based on a boric acid ester was synthesized.
5× Tris Borate EDTA (TBE) was made from TRIZMA Base and boric acid purchased from Sigma (St . Louis MO), and 0.5 M EDTA (OmniPur; Gibbstown, NJ).
5 µl of each sample was mixed with 1 µl loading dye and loaded onto a roti-safe (Carl Roth GmbH, Karlsruhe, Germany) stained 1 % agarose gel in tris base boric acid EDTA buffer (TBE; containing per liter 10.8 g tris base, 5.5 g boric acid, 20 mM EDTA) and migrated for approximately 1 h at 90 V.
Amplification products were electrophoresed on 1.3% agarose gels in TBE buffer (Tris base 87 mM, boric acid 89 mM, EDTA 2 mM, pH 8.0) in the presence of 0.5 μg/ml of ethidium bromide.
Finally, each PCR product was separated by gel electrophoresis on a 3% agarose gel buffered in 10.8 g/L Tris-base, 5.5 g/L boric acid, and 4 mL (vol/vol) 0.5 M EDTA, pH 8.0.
After denaturing at 95°C for 5 minutes, the DNA samples were placed on ice immediately and loaded onto precast 20% TBE acrylamide gels (Tris Base, Boric Acid, EDTA, 4% glycerol, and 20% acrylamide, Invitrogen, Carlsbad, CA).
Products from the purification were analyzed by electrophoresis on denaturing gels (6% acrylamide/bisacrylamide [19∶1]; 7.0 M urea; 1X TBE: 100 mM Tris-base, 83 mM boric acid, 1.0 mM EDTA).
PCR amplicons were analyzed on 3%55% High Resolution Blend agarose (Amresco) containing 0.20 µg/µl ethidium bromide in 1X TBE buffer (0.089 M Tris base, 0.089 M boric acid, and 2 m M ethylene-diaminetetraacetic acid).
Make a 10× stock solution: 0.89 M Tris base, 0.89 M boric acid, 20 mM EDTA pH 8.0.
Products were visualized on a 1% agarose gel stained with ethidium bromide in 0.5× TBE buffer (45 mM Tris base, 45 mM boric acid, and 1 mM EDTA; pH 8.0).
