. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. Figure 2. Spectrophotometnc scanning analysis of isolated total RNA from Bolrvllus schlmseri. (A) RNA isolated from B schlosseri with a conventional extraction method that utilizes phenol/chloroform/gua- nidme isothiocyanate (Chomcynski and Sacchi, 1987) demonstrates an altered ultraviolet absorption spectrum. In contrast (B), RNA isolated with the single-step cesium-chlonde method is free of contaminants and exhibits an optimal OD26o/OD280 ratio (). a cesium chloride ultracentrifugation step permitted the recovery of
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. Figure 2. Spectrophotometnc scanning analysis of isolated total RNA from Bolrvllus schlmseri. (A) RNA isolated from B schlosseri with a conventional extraction method that utilizes phenol/chloroform/gua- nidme isothiocyanate (Chomcynski and Sacchi, 1987) demonstrates an altered ultraviolet absorption spectrum. In contrast (B), RNA isolated with the single-step cesium-chlonde method is free of contaminants and exhibits an optimal OD26o/OD280 ratio (). a cesium chloride ultracentrifugation step permitted the recovery of high yields of spectrophotometrically and electrophoretically pure RNA preparations, irrespective of pigmentation or species. Groppe and Morse (1993) re- cently described a two-step cold method of isolating RNA from Haliotis ntfescens (red abalone); the method pro- vided high yields of pigment-free, undegraded material suitable for cDNA cloning. The first step, a phenol/chlo- roform extraction performed at 0°C, was crucial for the removal of ribonuclease activity, and the second step, employing ultracentrifugation through a cesium chloride gradient, removed an inhibitor of reverse transcriptase. The observations reported herein indicate that in Botryllus and other colonial ascidians, only a single preparative ul- tracentrifugation step through cesium chloride is required for isolation of biologically functional RNA. However, our findings are in contrast with those of Ku- mar el al. (1988), who reported that they successfully iso- lated RNA from various ascidians by using only a phenol/ chloroform-based procedure. In our hands, all prepara- tions isolated using phenol/chloroform were significantly contaminated with pigments and gave very poor yields. Furthermore, much of the original sample was left in the loading well during formaldhyde/agarose gel electropho- resis, and the efficiencies for in vitro translation reactions and reverse-transcription for cDNA library constructio
Size: 1512px × 1653px
Photo credit: © Library Book Collection / Alamy / Afripics
License: Licensed
Model Released: No
Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
