反应详情

温度

100 °C

Sodium bisulfite was prepared by adding 3.15 g of sodium sulfite and 7.15 g of sodium metabisulfite to 25 ml of water. 1,8-Diaminooctane (7.2 g) was added and the pH was adjusted to 7 with concentrated hydrochloric acid. Calf thymus DNA (Sigma, 139 mg) was dissolved in 20 mL of water and denatured by heating to 100° C. for 30 min followed by rapid cooling in an ice bath. The single stranded DNA was then sonicated for 40 min at 0° C. and added to the sodium bisulfite-diaminooctene solution. This gave a final concentration of 2 M bisulfite and 1 M diaminooctane. The mixture was clarified by centrifugation (4000×g) and then stirred at 60° C. (oil bath) for 42 hrs. The reaction mixture was cooled and filtered (0.2 μm filter) to remove a small amount of particulate material. The product was desalted in two 25 mL portions over a BioRad P-4 column (340×2.6 cm) equilibrated in 0.02 M sodium chloride, 1 mM EDTA, pH 8. After the void volume (60 mL), the product was collected in the next 50 mL fraction and each of these two fractions was separately dialyzed overnight against 4 L of water and lyphilized, yielding together 99.5 mg (72%). Seventy-five percent of the cytosine residues were substituted with DAO, based on alkaline hydrolysis-HPLC.

1. temperatureby rapid cooling in an ice bath
2. customwas then sonicated for 40 min at 0° C.