Experiment 9: Extraction and Identification of Yeast RNA 1. Objectives and requirements (1) Understand the method for extracting RNA from yeast. (2) Learn the identification methods for RNA components. (3) Learn and master the use of a centrifuge
2. Principles Microorganisms are raw materials for the industrial production of nucleic acids, with yeast being particularly ideal. This is because yeast nucleic acid mainly consists of RNA (2.67-10.0%), with very little DNA (0.03-0.514%). The yeast cells are easy to collect, and RNA is easily separated
2. Principles The commonly used methods in industrial production are the dilute alkali method and the concentrated salt method. - The former utilizes dilute alkali to dissolve the cell wall. This method has a short extraction time, but RNA is unstable under these conditions and prone to decomposition. - The latter involves using high-concentration salt to alter cell membrane permeability under heating conditions. This method is easy to master and produces a better product color
2. Principles To avoid molecular degradation, the phenol method can be used for RNA preparation. The principle is to treat biological materials with phenol to denature proteins, followed by centrifugation. The upper aqueous layer contains all the RNA, which can be precipitated with ethanol
2. Principles In this experiment, the dilute alkali method is used to extract RNA from yeast. The dilute alkali solution can dissolve the cell wall and release RNA, which dissolves in the dilute alkali solution. After neutralizing the alkali, ethanol can be added to precipitate the RNA, thereby obtaining crude RNA
2. Principles Attention should be paid to temperature during extraction to avoid prolonged exposure in the range of 20-70℃, as this is the temperature range where phosphodiesterases and phosphomonoesterases are active, which can degrade RNA and prevent its collection. Rapid heating to 90-100℃ to denature proteins and destroy these enzymes will be beneficial for RNA extraction
3.Methods and steps 3.1 Extraction of RNA (1) Weigh 6 g of dry yeast on a balance and place it in a 100 mL beaker. (2) Add 45 mL of 0.2% sodium hydroxide solution (NaOH), heat in boiling water for 20 min, and stir frequently. (3) Then, add about 7 drops of acetic acid to make the extraction solution slightly acidic (test with litmus paper)
3. Methods and steps 3.1 Extraction of RNA (4) Centrifuge for 10 min at 3500 rpm. (5) Take the supernatant, and while stirring, slowly add twice the volume of 95% ethanol to precipitate the RNA. After addition, let it stand for 10 min until complete precipitation. (6) Centrifuge for 3 min, pour the supernatant into a recovery bottle, and wash the precipitate twice with 95% ethanol. Each time you wash, carefully stir the precipitate with a fine glass rod, then centrifuge for separation. Each centrifugation should last for 3 min at 3500 rpm, and the supernatant should be poured into the corresponding recovery bottle
6 g yeast + 40 mL 0.2% NaOH boiling 20 min stirring Add 4-7 drops of acetic acid to adjust the pH until the blue litmus paper turns red 3500 rpm 10 min Add 2 volumes of ethanol to the supernatant to precipitate RNA Stay at RT for 5 min pellet + 10 mL ethanol stirring 3500 rpm, 3min pellet + 5 mL ethanol stirring 3500 rpm, 3min pellet + 5 mL ethanol Crude RNA
3.2 Identification of RNA Take 1/2 of the crude RNA product obtained above, add 5 mL of 10% sulfuric acid solution, and heat to boiling for 1-2 min to hydrolyze the RNA. • (1) Take 0.5 mL of the hydrolyzed solution, add 1 mL of orcinol-FeCl3 reagent, heat to boiling for 1 min, and observe the color change. • (2) Take 1 mL of 5% silver nitrate solution, add ammonia until the precipitate just disappears, and then gradually add the hydrolyzed solution drop by drop. Observe whether flocculent purine silver compounds are produced (sometimes the floccules appear slowly and can be left for more than ten min)