the education and research office of biochemistry and molecular biology yeast rna extraction and...

The Education and Research Office of Biochemistry and Molecular Biology

Yeast RNA extraction and component identification (strong salt method)


AimsAims Learn the procedure and principle of strong salt RNA

extraction method

Understand the procedure and principle of RNA

component identification


Principle Classification and distribution of nucleic acids

DNA ： Primarily locate in the nucleus; also exist in

plasmid.

RNA ： locate in ’cytoplasm


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


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：


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)


RNA component identification

Hy‘drolysis of RNA by boiling strong acid :

H2SO4 ( concentrated sulphuric acid)

RNA + H2O Base + Ribose + Phosphate boiling water bath


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


2. Base (purine) identification

(excess)(excess)

purine

Boiling water bath

Purine silver salt (brown)

aqueous am‘moniasilver nitrate


3. Ribose identification

1,3-di’hydroxy-5-’methylbenzene Cyan

furaldehyde

Concentrated HCl(Hydrochloric acid )


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


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


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


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 ？？


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


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


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.


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


Results

1 2 31 2 3 1 ： Phosphate identification (blue)

2 ： base identification (brown)

3 ： ribose identification (green)


Assignment questionsWhy 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?

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