Adenine → Sorbitol

Participants

Reaction Steps

Yeast (Zygosaccharomyces rouxii (obtained form Institute for Fermentation)) was inoculated in 5 ml of YPAD liquid culture medium (containing 10 g/liter of yeast extract, 20 g/liter of polypeptone, 0.4 g/liter of adenine and 20 g/liter of glucose), and was cultivated at 30° C. for 24 hours. The culture solution was centrifuged at 6,900×g for 3 minutes at 20° C. to collect the cells (hereinafter, the cells were collected under the same conditions). The obtained cells were suspended in 2 ml of a solution comprising 0.2-M Tris-HCl (pH 8.0) and 5 v/v % mercaptoethanol. The cell suspension was allowed to stand at 25° C. for 30 minutes while being gently stirred sometimes, and cells were then collected. Further, the collected cells were suspended 1 ml of a solution (pH 6.8) containing 2.5 mg/ml of Zaimolyeis-20T (obtained from SEIKAGAKU CORPORATION), 10 w/v % sorbitol and 5 w/v % KPO4. The suspension was allowed to stand at 30° C. for 90 minutes, and recentrifuged to collect the cells again. The collected cells were resuspended in 1 ml of a solution containing 0.2-M NaCl, 0.1-M EDTA, 5 w/v % SDS, 50-mM Tris-HCl (pH 8.5), and 20 Proteinase K were added to be 20 mg/ml thereto. The mixed solution was allowed to stand at 60° C. for 1 hour, then returned to room temperature, and extracted with a mixture of phenol and chloroform and then with chloroform to purify. To the thus-obtained supernatant was added in an equal volume of isopropanol to precipitate chromosomal DNA and plasmid pSR1. The mixture was centrifuged at 6,900×g for 10 minutes at 4° C. to collect the DNA, and the collected DNA was washed with 70 v/v % ethanol, then vacuum-dried and dissolved in 100 μl of TE.

Provenance & Evidence