Preparation of yeast tRNA by phenol method
Preparation of yeast tRNA by phenol method
By purification on DEAE cellulose columns, small amounts of DNA, large molecular RNA, proteins, polysaccharides and other impurities were removed, and finally a mixture of various amino acid-specific tRNAs was obtained.
Operation method
Preparation of yeast tRNA by phenol method
Materials and Instruments
DEAE Cellulose Diethanolamine (TEA) solution 0.lmol LTEA buffer 0.lmol L Sodium Chloride containing 0.lmoi. LTEA buffer 12mol L Sodium Chloride containing 0.lmol LTEA buffer lmol L Sodium Chloride containing 0.lmol LTEA buffer Ether Water saturated phenol Ethanol Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
Saponite Electric stirrer Centrifuge Partial collector UV spectrophotometer
1) DEAE cellulose: Weigh 20 g of l.Ommol/g DEAE cellulose, soak it in 300 ml of 0.5mol/L sodium hydroxide for 1~2 h with stirring, pour off the supernatant and then rinse it in water until neutral.
2) Diethanolamine (TEA) solution: firstly, adjust to pH7.4 with concentrated HC1, and then dilute to lmol/L as storage solution.
3) 0.lmol/L TEA buffer (pH7.4)
4) 0.lmol/L sodium chloride with 0.lmoi./LTEA buffer (pH 7.4).
5)12mol/L sodium chloride containing 0.lmol/LTEA buffer, pH 7.4.
6)lmol/L sodium chloride containing 0.lmol/LTEA buffer, pH 7.4.
7) Ether.
8) Water-saturated phenol.
9) Ethanol.
10) Soap Clay
11) Electric stirrer.
12) Centrifuge.
13) Partial Collector.
14) UV Spectrophotometer.
II Methods of operation
1) 250 g of fresh baker's yeast, add 375 ml of distilled water and stir well to form a paste.
2) Add 500 ml of 90% phenol (to change the cell permeability and denaturation of protein), stirring electrically for lh, and place in refrigerator overnight.
3) Centrifuge at 4000r/min for 15 min, discard the supernatant, the lower layer is the phenol phase, carefully aspirate (avoid inhaling proteins), add 20 g of potassium acetate and 2L of ethanol per liter to precipitate the tRNA, and leave it at -20°C overnight.
4) Centrifuge at 4000r/nin for 15 min, discard the supernatant, wash the precipitate with 95% ethanol, and centrifuge at 4000r./min for 15 min.
5) The precipitate was washed again with ether and centrifuged at 4000r/min for 15 min.
6) Dissolve the precipitate in 25 ml of 0.1mol/LTEA buffer. Add 0.025 g of saponite, 1.5 g of sodium chloride, leave at 0°C for lh and centrifuge at 4000r/min for 15 min.
7) The supernatant was diluted 10-fold with 0.1mol/I.TEA buffer, and the sample was loaded onto a DEAE cellulose column equilibrated with 0.lmol/LTEA buffer containing 0.lmol/LNaCl. The column was washed with TEA containing 0.2mol/LNaCl until the OD260 of the effluent was <0.05. The sample was diluted 10 times with TEA buffer.
8) Elute with TEA containing 1mol/LMaCl, collect in portions, measure the OD260, and take the peak portion of the liquid.
9) Add 2 times volume of 95% ethanol, overnight at 20℃ (or 2 h).
10) centrifuge at 4000r/min for 15 min, discard the supernatant, and wash the precipitate with 67% ethanol, 95% ethanol, anhydrous ethanol and ether.
11) Centrifuge at 4000r/mim, dry the precipitate under vacuum, and electrophoresis for tRNA detection.