The Education and Research Office of Biochemistry and Molecular Biology Yeast RNA extraction and component identification (strong salt method)

The Education and Research Office of Biochemistry and Molecular Biology Aims Learn the procedure and principle of strong salt RNA extraction method Understand the procedure and principle of RNA component identification

The Education and Research Office of Biochemistry and Molecular Biology Principle Classification and distribution of nucleic acids  DNA ： Primarily locate in the nucleus; also exist in plasmid.  RNA ： locate in ’cytoplasm

The Education and Research Office of Biochemistry and Molecular Biology The choice of sample ： Yeast RNA （ The weight of RNA accounts for 3%-10% of the total dry weight. ） Extraction methods ： dilute ’alkali method, strong salt method

The Education and Research Office of Biochemistry and Molecular Biology Cell lysis Extraction Purification I. Material preparation II. Lyse cell and release cellular contents III. Nucleic acid separation and purification IV. Precipitate nucleic acid and remove impurities V. Dissolve nucleic acid in buffer or water Procedure ：

The Education and Research Office of Biochemistry and Molecular Biology RNA dissociates as a soluble sodium salt Centrifuge to remove cell debris and collect supernatant; adjust the pH to the RNA isoelectric point （ pH 2.0) Precipitate RNA and collect them by centrifugation Wash precipitation to remove impurity and purify RNA Boil yeast in strong salt solution to lyse the cells and pre’cipitate denatured proteins RNA extraction (strong salt method)

The Education and Research Office of Biochemistry and Molecular Biology RNA component identification Hy‘drolysis of RNA by boiling strong acid : H 2 SO 4 ( concentrated sulphuric acid) RNA + H 2 O Base + Ribose + Phosphate boiling water bath

The Education and Research Office of Biochemistry and Molecular Biology 1. Phosphate identification Identify RNA with its three hydrolytic products: Phosphomolybdic acid+FeSO 4 molybdenum blue ammonium molybdate Phosphomo‘lybdic acid (PMA) ferrisusfas

The Education and Research Office of Biochemistry and Molecular Biology 2. Base (purine) identification (excess) purine Boiling water bath Purine silver salt (brown) aqueous am‘monia silver nitrate

The Education and Research Office of Biochemistry and Molecular Biology 3. Ribose identification 1,3-di’hydroxy-5-’methylbenzene Cyan furaldehyde Concentrated HCl(Hydrochloric acid )

The Education and Research Office of Biochemistry and Molecular Biology Instruments and reagents instruments Precision pH test strips (pH 0.5 ～ 5.0), universal pH test strip (pH 1 ～ 14), flasks, centrifuge, balance, electric stove 10 ％ NaCl 、 6M HCl 、 5 ％ ’aqueous am‘monia, (NH 4 ) 2 MoO 4, 1,3-dihydroxy-5-methylbenzene, 0.1M AgNO 3, FeSO 4, 1.5M H 2 SO 4, concentrated ammonia, Fe 3+ ·HCL reagents

The Education and Research Office of Biochemistry and Molecular Biology Procedure  Yeast RNA extraction (4 persons in 1 group) 1. Yeast lysis ： 10 ％ NaCl(40mL) 100mL flask boil Add dry yeast (one spoon) Boiling water bath for 40min Cool down Mix with glass rod

The Education and Research Office of Biochemistry and Molecular Biology Take out the flask Cool down with tap water and transfer into 50mL centrifugation tube 3500rpm for 5min Collect supernatant 2.Centrifugation for RNA separation Balance tubes before centrifugation

The Education and Research Office of Biochemistry and Molecular Biology 3. Purify RNA by precipitation Transfer supernatant into a small flask Cool on ice bath Adjust pH to 2.0 ～ 2.5 (RNA isoelectric point) with 6M HCL Stand in ice bath for 5- 10min (precipitation shows up at the bottom) Transfer to centrifuge tube after gentle shaking, and balance before centrifugation 3500rpm for 5min ， and collect precipitation Precise pH test trips  How to adjust pH ？

The Education and Research Office of Biochemistry and Molecular Biology  RNA component identification 1.Dissolve RNA 2. RNA hydrolysis (2 person in 1 group from here) Add 22 drops 1.5M H 2 SO 4 Boiling water bath 10min Transfer 5ml RNA to large test tube clear solution Add 1 drop distilled water mix to slurry form add 10ml distilled water and mix add 5 ％ aqueous ammonia till pH 6-7, continue add aqueous ammonia till precipitation dissolves Universal pH test trips

The Education and Research Office of Biochemistry and Molecular Biology Purine identification ： 20 drops hydrolysate+20 drops 0.1mol AgNO 3 Boiling water bath 15 min Ribose identification ： 20 drops hydrolysate+20 drops Fe 3+ ·HCL+ 2 drops 5% 1,3-dihydroxy-5-methyl'benzene 3. RNA component identification Phosphate identification: 20 drops hydrolysate+20 drops ammonium molybdate +FeSO 4 crystals Add concentrated ammonia till precipitation is gone +10 drops concentrated ammonia Boiling water bath 2-3 min mix Boiling water bath 2 min ？ ？ ？ (sesame size) ferrisusfas

The Education and Research Office of Biochemistry and Molecular Biology 4. Application of centrifuge centrifuge is a technique using the centrifugal force and the sedimentation principle to separate and concentrate substance. It is a necessary tool of separation and purification in biochemistry, molecular biology, cell biology, genetics, chemistry and food industry.

The Education and Research Office of Biochemistry and Molecular Biology 5.Centrifuge operation 1.choose proper tubes 2.balance tubes (including the sheath) 3.turn on power and set rpm and time 4.press “ 停止 ” button to open the lid; place tubes in a central symmetric way 5.close lid tightly and press “ 离心 ” to start centrifugation 6.Open the lid only when the rotor has fully stopped

The Education and Research Office of Biochemistry and Molecular Biology Results ： Phosphate identification (blue) 2 ： base identification (brown) 3 ： ribose identification (green)

The Education and Research Office of Biochemistry and Molecular Biology Assignment questions  Why do you put the flask with yeast and 10% NaCl in the water bath for cell lysis after water is boiling?  What are the purposes of the twice pH adjustment in this experiment?