A detergent-citric acid technique for isolating nuclear and cytoplasmic fractions containing undegraded RNA from cells of Xenopus laevis

✍ Scribed by Leo Miller

• Publisher: Elsevier Science
• Year: 1979
• Tongue: English
• Weight: 722 KB
• Volume: 100
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(79)90127-1

✦ Synopsis

A simple technique is described for fractionating cells ofxenopus laevis using Triton X-100 and citric acid. Cells are treated for 1 min with a hypotonic buffer containing 0.5% Triton X-100 and RNAase inhibitors. The cells are then disrupted by adding citric acid to a final concentration of 1% for 2 min and the nuclei are pelleted by centrifugation. The detergentcitric acid fractionation technique has been used to obtain pure nuclear and cytoplasmic fractions containing undegraded RNA from an epithelial cell line of Xenopus laevis. The procedure is very gentle, eliminates RNAase activity, and can be performed in about 5 min. The detergent-citric acid technique can be easily scaled up or down and has also been used successfully with mammalian and insect cell lines.

MATERIALS AND METHODS

Cell Lines

An epithelial cell line (X1-177) derived from swimming tadpoles of Xenopus laevis in January, 1977, was used throughout this study. The cells were maintained in 70% Leibovitz medium (L-15) containing 10% fetal calf serum and 100 E.cg/ml Gentamicin