Formamide → Fluorescein 5-isothiocyanate

Participants

Reaction Steps

5× SSC (0.75M NACl, 0.075M sodium citrate); 30% Formamide (v/v); 3% Triton X-100 (v/v) (Triton X-100 is an alcohol derivative of polyoxyethylene ether, see Aldrich Chemical Co. catalogue for 1990-91); 0.4M Guanidine isothiocyanate; 0.16M sodium phosphate buffer (pH 6); 15× Ficoll/PVP (polysucrose 400,000 mol wt/polyvinyl pyrrolidone); 1 mg/ml Sheared Salmon Sperm DNA; 10 mM EDTA; 25 mM DTT; 5% PEG 4000. In the foregoing, 500× Ficoll/PVP is 5 g of Ficoll type 400 (polysucrose 400,00 mol wt) plus 5 g of PVP (polyvinylpyrrolidone) dissolved in water to a total volume of 100 ml; 15× Ficoll/PVP indicates that 500× Ficoll/PVP has been diluted by a factor of 15/500 with water. Ficoll (Pharmacia) is a nonionic synthetic polymer of sucrose. A stock solution of 0.1 mg/ml FITC in dimethylformamide was prepared. It was added to the HC in a quantity to produce a final concentration of FITC in the HC of 2 μg/ml.

Provenance & Evidence