Purification of rRNA from Ribonucleoprotein (RNP)
Complexes
Ribosomes and ribosomal subunits were manually collected from sucrose
gradients as peak fractions. The sucrose fractions were collected into
15 mL falcon tubes and diluted at least two-fold with the gradient
buffer (25 mM Tris-HCl pH 7.9, 100 mM KCl, 10 mM MgCl2).
Next, 2.5 vol. of 96% ethanol was added to the samples and incubated at
-20°C overnight. The fractions were pelleted via centrifugation for 45
minutes at 4000 rpm +4°C (4K15 Sigma, rotor 11150). The pellet was
washed with 70% EtOH, and centrifugation was re-applied for 10 minutes.
The ribonucleoprotein complexes were suspended in 0.1 mL of MilliQ
water, and samples were stored at -20°C.
The rRNA was purified with phenol-chloroform extraction. The samples
were kept on ice, and 1% SDS-containing phenol was added to the
samples. Samples were vortexed vigorously for 10 s, kept on ice for 5
min, and centrifuged at 16,100xg 10 min at +4°C. The water phase was
transferred to a new microfuge tube into which chloroform:phenol mixture
(1:1) was added and vortexed for 10 seconds, centrifuged as above. This
step was repeated, using only chloroform to avoid phenol carryover. The
water phase was transferred to a new microfuge tube, and the RNA was
precipitated with 0.1 vol. 3M sodium acetate (pH 5.2-5.5) and 2.5 vol.
ethanol at -20°C for 1 hour. The pellet was washed with 70% EtOH and
dried at room temperature for 5 minutes. The purified RNA was dissolved
in ultra-pure distilled water (MilliQ).