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

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

3. 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
mitochondria, chloroplasts, and plasmid.
 RNA：locate in cytoplasm

4. 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
 reagents
10％NaCl、6M HCl、5％aqueous ammonia,
(NH4)2MoO4, 1,3-dihydroxy-5-methylbenzene, 0.1M AgNO3,
FeSO4, 1.5M H2SO4, concentrated ammonia, Fe3+·HCL

5. 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

6. 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：

7. The Education and Research Office of Biochemistry and Molecular Biology
 RNA extraction (strong salt method)
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～
2.5）
Precipitate RNA
and collect them
by centrifugation
Boil yeast in strong salt
solution to lyse the cells
and precipitate
denatured proteins
Wash precipitation
to remove impurity
and purify RNA
Cool the flask
with flowing water

8. The Education and Research Office of Biochemistry and Molecular Biology
 RNA component identification
Hydrolysis of RNA by boiling strong acid :
H2SO4
RNA + H2O Base + Ribose + Phosphate
boiling water bath

9. The Education and Research Office of Biochemistry and Molecular Biology
Identify RNA with its three hydrolytic
products:
1. Phosphate identification
aammmmoonniiuumm mmoollyybbddaattee
PPhhoosspphhoommoollyybbddiicc aacciidd ((PPMMAA))
PPhhoosspphhoommoollyybbddiicc aacciidd++FFeeSSOO44 mmoollyybbddeennuumm bblluuee

10. The Education and Research Office of Biochemistry and Molecular Biology
2. Base (purine) identification
((eexxcceessss))
purine
Boiling
water bath
Silver ammonia complexes
Purine silver salt (brown)

11. The Education and Research Office of Biochemistry and Molecular Biology
3. Ribose identification
furaldehyde
Concentrated HCl
Boiling
water bath
1,3-dihydroxy-5-methylbenzene Cyan

12. The Education and Research Office of Biochemistry and Molecular Biology
Procedure
 Yeast RNA extraction (in 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

13. The Education and Research Office of Biochemistry and Molecular Biology
2. Centrifugation for RNA separation
Take out the
flask
Cool down with flowing
water and transfer into
50mL centrifugation tube
3500rpm
for 10min
Collect
supernatant
Balance tubes
before
centrifugation
Boil yeast in
strong salt
solution

14. 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
5~10min，and
collect
precipitation
Precise pH
test trips
HHooww ttoo aaddjjuusstt ppHH ？？
6-7 drops of HCl

15. The Education and Research Office of Biochemistry and Molecular Biology
 RNA component
1.Dissolve RNidAentification
Add 1 drop distilled water mix to slurry form add 10ml distilled
water and mix add 5％aqueous ammonia till pH 8, continue add aqueous
ammonia till precipitation dissolves
2. RNA hydrolysis
Add 22 drops 1.5M H2SO4
Transfer 5ml RNA to large test tube clear solution
Boiling water bath 10min
Universal pH
test trips

16. The Education and Research Office of Biochemistry and Molecular Biology
3. RNA component identification
Add concentrated
ammonia till
precipitation is gone
Purine identification ：
20 drops hydrolysate+20 drops 0.1mol AgNO3
Boiling
water bath
15 min
+10 drops
concentrated
？？
ammonia
？？
？？ (sesame size)
Ribose identification ：
20 drops hydrolysate+20 drops Fe3+·HCl+ 2 drops 5% 1,3-dihydroxy-5-methylbenzene
Phosphate identification:
20 drops hydrolysate+20 drops ammonium molybdate +FeSO4 crystals
Boiling
water bath
2-3 min
mix
Boiling
water bath
2 min

17. 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, pharmaceutics and food
industry.

18. 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

19. The Education and Research Office of Biochemistry and Molecular Biology
Results
11 22 33 1：ribose identification
(cyan)
2：base identification
(brown)
3：Phosphate
identification (blue)

20. 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 first pH adjustment in this
experiment?