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
AimsAims Learn the procedure and principle of strong salt RNA
extraction method
Understand the procedure and principle of RNA
component identification
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Principle Classification and distribution of nucleic acids
DNA : Primarily locate in the nucleus; also exist in
plasmid.
RNA : locate in ’cytoplasm
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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:
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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 :
H2SO4 ( concentrated sulphuric acid)
RNA + H2O 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 acidPhosphomolybdic acid+FeSO+FeSO44 molybdenum bluemolybdenum blue
ammonium molybdateammonium molybdate
Phosphomo‘lybdic acid Phosphomo‘lybdic acid (PMA)(PMA)
ferrisusfas
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2. Base (purine) identification
(excess)(excess)
purine
Boiling water bath
Purine silver salt (brown)
aqueous am‘moniasilver nitrate
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3. Ribose identification
1,3-di’hydroxy-5-’methylbenzene Cyan
furaldehyde
Concentrated HCl(Hydrochloric acid )
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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)2MoO4, 1,3-dihydroxy-5-methylbenzene, 0.1M AgNO3,
FeSO4, 1.5M H2SO4, concentrated ammonia, Fe3+·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
boilAdd dry yeast (one
spoon)
Boiling water bath for 40min
Cool downMix with glass rod
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Take out the flask
Cool down with tap water and transfer
into 50mL centrifugation tube
3500rpmfor 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 How to adjust pH ??
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RNA component identification
1.Dissolve RNA
2. RNA hydrolysis (2 person in 1 group from here)Add 22 drops 1.5M H2SO4
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
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Purine identification :20 drops hydrolysate+20 drops 0.1mol AgNO3
Boiling water bath
15 min
Ribose identification :20 drops hydrolysate+20 drops Fe3+·HCL+ 2 drops 5% 1,3-dihydroxy-5-methyl'benzene
3. RNA component identification
Phosphate identification:
20 drops hydrolysate+20 drops ammonium molybdate +FeSO4 crystals
Add concentrated ammonia till
precipitation is gone+10 drops
concentrated ammonia
Boiling water bath
2-3 min
mix Boiling water bath2 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.
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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
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Results
1 2 31 2 3 1 : Phosphate identification (blue)
2 : base identification (brown)
3 : ribose identification (green)