Cool down the gel at room temperature and poor it to a gel
chamber with appropriate combs until complete solidification.- When the gel is ready, place it in an agarose gel electrophoresis
chamber, and then fill the chamber with 1TBE until the gel
surface is covered. - In a 1.5 mL RNase-free microtube, prepare a mix containing
1.3μL of yeast total RNA with 5μL of gel loading dye and
33.7μL of RNase-free water. - Load 10μL of the mix into 4 adjacent wells on the gel.
- Connect the gel electrophoresis chamber to a power supply and
run electrophoresis at constant 60 V for 4–5 h. - When the electrophoresis is over, cut from top to bottom the
two external lanes surrounding the central lanes of the gel with
a clean scalpel and stain them in 50 mL of 1TBE containing
5 μL of SYBR Gold dye for 15 min under gentle shaking. - Locate the bands of interest (18S and 25S rRNA) using a long-
wavelength UV-lamp (λ¼365 nm) and excise those two bands
out from the gel with a scalpel. - Reconstitute the entire gel and cut the corresponding gel slices
containing the RNA fragments of interest from the nonstained
middle lanes of the gel with a clean and sharp scalpel blade. - Place the gel slices in separate RNase-free 1.5 mL tubes and
weight them. - Elute the RNAs from the gel in an equal volume (w/v) of
RNase-free water. Melt the agarose gel by heating until com-
plete gel dissolution (takes about 30 min). - Proceed to phenol extraction (seeNote 7) followed by a chlo-
roform extraction step. Precipitate eluted RNA by the addition
of 1/10th volume of 3 M sodium acetate and 3 volumes of 96%
ethanol for 15 min at 80 C followed by a centrifugation step
of 15 min at 12,000–16,000gin a refrigerated tabletop
centrifuge. - Wash the pellet with 80% ethanol, dry and dissolve it in 20μL
of RNase-free water. - Quantify the 18S and 25S rRNA by measuring A260nmusing a
UV-spectrophotometer Check the quality of the purified RNA
by using the Agilent 2100 Bioanalyzer (seeSubheading3.4)
and proceed to yeast rRNA reconstruction (Fig.1).
3.5 Yeast rRNA
Reconstruction
- Mix equimolar amounts of 18S and 25S in vitro RNA tran-
scripts (seeNote 8) in a 1.5 mL Eppendorf tube to get 100 ng
at a final concentration of 10 ng/μL. - Follow the same procedure with purified 18S and 25S RNA.
38 Lilia Ayadi et al.