Reaction, with bromphenol blue

Chen and Laidler [Canadian J. Chem., 37 (599), 1959] have studied the reaction of the quinoid form of bromphenol blue with hydroxide ions to give the carbinol form of the dye. The following values of the second-order rate constant at 25 °C were reported. 

The deprotected oligonucleotide synthetic product is precipitated twice in ethanol, and a 0.5 fig/fd solution in water is prepared (concentration is measured from a UV absorption spectrum). One microliter of the oligo-deoxynucleotide solution is mixed with 2 fd of 10X PL, 5 fd of [y-32P]ATP (or [y-35S]ATP), 1 fd of T4 polynucleotide kinase, and 11 fd water. After incubation at 37 ° (for 45 min with [y-32P]ATP or for 2 hr with [y-35S]ATP), the reaction is stopped by the addition of 150 [A of 5 M ammonium acetate, pH 5.5, and 130 fd water and 10 fd of the yeast tRNA solution are added to the mixture before precipitation with 1 ml ethanol. After chilling at —70° for at least 15 min, the precipitate is collected by centrifugation (12,000 g, 15 min), redissolved, and submitted to two additional cycles of precipitation-redissolution. Finally, the precipitate is redissolved in 20 fd of gel loading mix and the mixture analyzed on a 8% acrylamide-7 Af urea slab gel in IX electrophoresis buffer, until the bromphenol blue has reached the middle of the gel. 

Twenty fmol of 5 -radiolabeled single-stranded or double-stranded oligonucleotides is incubated with 1.3 pmol of the Rep protein (protein to DNA molar ratio of 60 1) for 5 min. The reaction is stopped by addition of 25 mM EDTA and 5 p.1 of loading buffer (30% glycerol, 0.25% xylene cyanol, and 0.25% bromphenol blue). The products are then loaded on a 8% polyacrylamide gel (30 1) in 5% glycerol, 0.25 x TBE, and run in 0.25 x TBE at room temperature. Incubation temperature is not important for migration under native conditions, and no differences are observed when protein and DNA are previously incubated together either at 105° and 4°. 

Bromide Reaction of h q)obromite with phenol red to form bromphenol blue-type indicator... 

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