Extraction and isolation of rat liver rRNAs
Rat liver was homogenized, the supernatant was centrifuged, and RNA was extracted with a mixture of phenol and m-cresol. rRNA and mRNA were separated in the presence of naphthalene 1,5-disulfonic acid, and pure rRNA was obtained.
Operation method
Extraction and isolation of rat liver rRNAs
Materials and Instruments
Rat liver Phenol-cresol mixture Naphthalene 1.5-disulfonic acid solution Wash solution A Wash solution B Ethanol solution Triisopropyl naphthalene sulfonic acid sodium salt Anhydrous ethanol Move Materials and equipment For more product details, please visit Aladdin Scientific website.
1) rat liver: rats were killed by neck beating, the liver was taken out and immediately placed in liquid nitrogen plants for spare use
2) phenol - cresol mixture: 500 g of phenol (must be recrystallized) and 70 mim-cresol were dissolved in 50 m l of distilled water and 0.5 g of 8-hydroxyquinoline was added
3) naphthalene 1.5-disulphonic acid solution: 0.5 g of naphthalene 1.5-disulphonic acid was dissolved in 100 ml distilled water 4) washing solution A: 20 g of sodium benzoate, 10 ml of m- cresol and 3 g of sodium chloride were dissolved in 100 ml distilled water 5) washing solution B: 75 ml of water-free ethanol and 3 g of sodium chloride were added
4) Wash solution A: 20 g of sodium benzoate, 10 ml of m- cresol and 3 g of sodium chloride in 100 ml of distilled water
5) Wash solution B: 75 ml of anhydrous ethanol and l g of sodium heliotrope in 25 ml of distilled water
6) 75.% ethanol solution
7) Sodium triisopropyl naphthalene sulfonate
8) Anhydrous ethanol
2) Methods of operation
1) Weigh 15 g of murine liver, and add it to 10 times the volume of a mixed phenol-cresol solution and to 10 times the volume of a solution of a mixture of phenol and cresol. phenol-cresol solution and 10 times volume of naphthalene 5-disulfonic acid solution. Homogenize with a homogenizer under
2) Remove the homogenate and place it in a beaker. Stir at 2°C for 20 min, centrifuge at 6000g at 50°C for 15 min, and discard the precipitate.
3) Add sodium triisopropylnaphthalenesulfonate to the upper extract to a final concentration of 5%, then add 1/2 the volume of phenol-cresol solution, stir at 20°C for 20 min, and then centrifuge at 8000g at 5°C for 10 min.
4) Mix the liver with a 10-fold volume of phenol-cresol mixture and 10-fold volume of naphthalene 5-disulfonic acid solution per 100 ml of centrifuge. ) To each 100 ml of centrifugation supernatant, 3 g of sodium chloride, 2 g of sodium benzoate and 10 mim-cresol were added sequentially. Stir to dissolve homogeneously, then centrifuge the supernatant at 8000g for 10 min at 5°C. Discard the supernatant.
5) Centrifuge and wash the precipitate in the following order: wash twice with cold Wash A, wash once with Wash B, wash once with 75% ethanol, and then wash twice with anhydrous ethanol, and then wash twice with anhydrous ethanol, and use 25 ml of each time to obtain the pure murine liver rRNA. Remove the rRNA, and place it in a vacuum desiccator equipped with CaCl2 to dry it.