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Typical density gradient fraction analysis after 36 h of centrifugation using 6.4 ml volume and 1.8 g ml-1 starting density CsTFA gradient on RNA extracted from soils amended with [14C]glucose at a concentration of 150 μg C g-1 soil after 4 days of incubation. Points are means of triplicate fractions. Fraction divisions indicate the buoyant density ranges for the “heavy” fractions containing 14C-labeled RNA (solid lines), “light” fractions containing 12C-unlabeled RNA (dotted lines), and “intermediate” fractions containing 14C/12C-labeled RNA (dashed lines).
Autoradiograph and plot of spot intensity showing distribution of 14C-labeled RNA over the first 20 density gradient fractions after 36 h of centrifugation. RNA was extracted from soils amended with [14C]glucose at a concentration of 150 μg C g-1 soil after 4 days of incubation. RNA from each fraction was spotted onto a membrane and exposed to X-ray film for 2 to 3 weeks. Arrows show the fraction ranges for the “heavy” fractions (3 to 7) containing 14C-labeled RNA, “light” fractions (15 to 17) containing 12C-unlabeled RNA, and “intermediate” fractions (11 to 14) containing 14C/12C-labeled RNA.
Distribution of 16S rRNA over the first 20 density gradient fractions after 36 h of centrifugation, estimated using real time qPCR. RNA was extracted from soils amended with [14C]glucose at a concentration of 150 μg C g-1 soil after 4 days of incubation. RNA from each fraction was reverse transcribed and quantified. Arrows show the fraction ranges for the “heavy” fractions (3 to 7) containing 14C-labeled RNA, “light” fractions (15 to 17) containing 12C-unlabeled RNA, and “intermediate” fractions (11 to 14) containing 14C/12C-labeled RNA.
RT-PCR-DGGE gels comparing the bacterial community profiles recovered from the first 20 fractions along the RNA-SIP density gradient after 36 h of centrifugation. RNA was extracted from soil in microcosms amended with different concentrations of [14C]glucose (50 μg C g-1 soil) after 4 days of incubation. The “heavy” fractions (3 to 7) containing the 14C-labeled RNA, the “light” fractions (15 to 17) containing the 12C-unlabeled RNA, and the “intermediate” fractions (11 to 14) containing 14C/12C-labeled RNA are shown. M indicates the marker lane, and numbers represent fraction number. Boxes indicate the bands that were excised from the gel, reamplified, and sequenced.
Histogram showing the relative band intensity for DGGE-derived 16S rRNA ribotypes recovered in both the “heavy” and “light” fractions. RNA was extracted from soil in microcosms amended with different concentrations of [14C]glucose (0, 15, 50, and 150 μg C g-1 soil) after 4 days of incubation. RT-PCR-DGGE comparing the bacterial community profiles was then performed on the first 20 fractions along the RNA-SIP density gradient after 36 h of centrifugation. The “heavy” fractions (3 to 7) containing the 14C-labeled RNA and the “light” fractions (15 to 17) containing the 12C-unlabeled RNA are shown. M indicates the marker lane, and numbers represent fraction number. Bands showing marked changes in intensity were excised from the gel, reamplified, and sequenced.
Relative band intensity and distribution of the DGGE 16S rRNA ribotypes over the first 20 fractions. DGGE 16S rRNA ribotypes found in the “heavy” fractions (3 to 7) contained the 14C-labeled RNA (black solid lines), while ribotypes found only in the “light” fractions (15 to 17) contained the 12C-unlabeled RNA (black dashed lines). RNA was extracted from soil microcosms amended with different concentrations of [14C]glucose (0, 15, 50, and 150 μg C g-1 soil) after 4 days of incubation. RT-PCR-DGGE comparing the bacterial community profiles was then performed on the first 20 fractions along the RNA-SIP density gradient after 36 h of centrifugation. Bands showing marked changes in intensity were excised from the gel, reamplified, and sequenced.
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