RNA Detection

8. 80% ethanol.


9. Mini Quick Spin RNA column (e.g., Roche).


10. UV spectrophotometer (e.g., NanoDrop 2000c).

2.3 Yeast Total RNA
Extraction

1. Yeast cell culture (5 mL of yeast culture grown to an OD 600
   of 5–9).


2. AE buffer: 50 mM NaOAc in water, pH 5.2, 10 mM EDTA.


3. 10 w/v % SDS.


4. RNase-free water.


5. 1.5 mL RNase-free microcentrifuge tubes.


6. Acid phenol, pH 4.5.


7. Phenol–chloroform–isoamyl alcohol mix (25:24:1, v/v).


8. Chloroform.


9. 3 M NaOAc in water, pH 5.2.


10. 96% ethanol.


11. 80% ethanol.


12. Dry ice.


13. Refrigerated tabletop centrifuge.


14. Water bath or heating block, set to 65C.

2.4 Purification of
18S and 25S rRNA
from Yeast Total RNA

1. Low Melting agarose (e.g., Lonza Nu Sieve®GTG®Agarose).


2. 10TBE buffer: 108 g Tris, 55 g boric acid, 9.3 g EDTA,
   pH 8, H 2 O qsp 1 L.


3. RNase-free water.


4. 1TBE buffer: 100 mL of 10TBE and 900 mL of RNase-
   free water.


5. Microwave oven.


6. Agarose gel electrophoresis chamber.


7. Electrophoresis power supply.


8. 3μg/μL yeast total RNA preparation.


9. 1.5 mL RNase-free microcentrifuge tubes.


10. 6DNA loading dye: 1.9 mM xylene cyanol, 1.5 mM bromo-
    phenol blue, and 25% glycerol.


11. SYBR®Gold Nucleic Acid Gel Stain 10,000concentrate in
    DMSO (Invitrogen).


12. UV 365 nm transilluminator.


13. Scalpel or razor blade.


14. Heat block preheated at 65C.


15. Ultrapure phenol buffered with Tris–HCl pH 8.0.

32 Lilia Ayadi et al.