International University, Vietnam National University - HCMC

GENETICS LABORATORY

GENETICS LABORATORY

REPORT 2: DNA EXTRACTION FROM PLANT CELLS

Group: 2 Section: Friday

Date: 15/10/2021

SUBMITDAY: 22/10/2021

INSTRUCTOR: TRAN HAI YEN

COURSE: BTBT19IU11
Group members:
Lê Thị Phương Thùy - BTBTIU19125
Dương Bảo Khôi - BTBTIU19072
Nguyễn Ngọc Hải My - BTBTUN19011
Trương Anh Thư - BTBTIU19121

Score: _________
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GENETICS LABORATORY

A. Introduction:

DNA (deoxyribonucleic acid), a molecule contains genetic instructions of living

organisms in order to develop and reproduce. We extract DNA for modification of
plants, isolate DNA with desirable traits then inject them into the genome of plants.
Principle of this experiment is to break down the membrane and go into the nucleus.
First stage is grinding the tissue in liquid nitrogen. Next is purifying DNA by
centrifugation to precipitate cell debris. After that, DNA must be precipitated and
washed. DNA can be resuspended in SDS and stored in sterile form. Then, the
presence of DNA in solution is observed under UV light with ethidium bromide.

B. Purpose:

- Understand the process of extracting DNA from plant cells.

- Identify extraction that contains DNA.

C. Procedure:
Sample preparation > Cell lysis > Protein removal > DNA precipitation > DNA
dehydration

Visualized procedure
1. Grind 200mg of plant tissue in 500ul of SDS buffers.
2.Transfer SDS/plant extract mixture to an Eppendorf.
3. Incubate the SDS/plant extract mixture for about 15min at 55ºC in a water bath.
_Spin 12000 g in 5min.
4. Transfer supernatant to a clean Eppendorf.
5. Add 500ul Chloroform: Isoamyl Alcohol (24:1) and mix the solution by inversion.
_Spin 13000rpm in 1min.
6. Transfer upper aqueous phase to clean Eppendorf.
7. Add 50ul 7.5M Ammonium Acetate. Invert the Eppendorf to mix.
8. Add 500ul of ice-cold absolute Ethanol. Invert Eppendorf several times.
_Incubate at -20ºC in 30-60mi.
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_Spin 13000rpm in 1min.

9. Remove the supernatant. Then wash the DNA pellet by 500ul ice cold 70% Ethanol.
_Spin 13000rpm in 1min.
10. Remove the supernatant.
(*) Note: Step 9,10 is done twice.
11. Dry DNA pellet in 15min. Do not allow the DNA to over dry.
12. Resuspend DNA in 50-400ul sterile DNase free water (additional 10ug/ml RNase
A).
_Incubate at 65ºC in 20min.
_Store at 4ºC.
13. Check the presence of DNA in the solution by using UV light and Ethidium
Bromide.

D. Result:

The DNA is now precipitated out of the solution. If there is a high or enough
amount of DNA, we will see the stringy white mass in the upper phase.

E. Discustion:
A. Question in lab manual:

1. Explain the function of SDS buffer?

• SDS which stands for 'Sodium Dodecyl Sulfate'. It is strong anionic

detergent that can solubilize the proteins and lipids that form the
membranes. It removes the negative ions from the protein and destroys its
confirmation. Because of loss of confirmation the protein loses its structure.
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GENETICS LABORATORY

The proteins from the cell membrane get damaged and cell gets broken.
This will help the cell membranes and nuclear envelopes to break down and
expose the chromosomes that contain the DNA. In addition to removing the
membrane barriers, SDS helps release the DNA from histones and other
DNA binding proteins by denaturing them.

2. Function of Ammonium acetate?

• Ammonium acetate is an ammonium salt obtained by reaction
of ammonia with acetic acid. A deliquescent white crystalline solid, it has a
relatively low melting point (114℃) for a salt. Used as a food acidity
regulator, although no longer approved for this purpose in the EU. It has a
role as a food acidity regulator and a buffer. It is an acetate salt and an
ammonium salt.

3. What is the colour of DNA after extraction?

• It looks like strands of mucus; limp, thin, white noodles; or a network of
delicate, limp fibers.

4. How do you know your extraction contain DNA?

• When you can see the strands, thin and white fibre in the extraction

B. The function of chemical and explain when using:

1. Grind plant tissue: this step is to separate the cells. It destroys the cell
membrane.

2. Liquid Nitrogen
Liquid nitrogen is used to freeze cell walls and produce fine grinds (the finer
the grind, the greater the yield) while keeping harmful chemicals and
cellular enzymes deactivated, thus reducing shear and damages to the
DNA. It also shuts down the activity of the enzyme in the cell.

3. Proteinase K and Incubation in 60-degree Celsius

Proteinase K, which is one type of enzyme. It destroys protein (degrade
protein)

After that, incubation at 60 degrees Celsius, helping Proteinase K to act as

highly effective (increase the effectiveness of active proteinase K). Each
Enzyme has a suitable temperature for its activity.

4. In step 7
● The upper phase contains : Has protein, DNA, RNA, maybe protein
DNA is a hydrophilic substance. Moreover, DNA has a higher density than
others, so it goes to the top of the solution.
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GENETICS LABORATORY

● Inter phase contains: foam, bubble (white color). When we centrifuge it

occurs the foaming reaction.

● The lowest phase contains: Chloroform and another (RNA, protein).

They are hydrophobic substances. They are pulled down.

5. The chloroform-isoamyl alcohol

The chloroform-isoamyl alcohol is to remove the proteins, most lipids, and
cellular debris that can cause an impure DNA result. It does so by binding
with non-aqueous compounds and separating them from the aqueous
compounds. Centrifuging this will create a precipitate of clumped proteins,
non-aqueous lipids, and cellular debris, whereas the DNA is extracted from
the supernatant.

Isoamyl alcohol reduces foaming between the interphase. If not using

isoamyl alcohol to reduce air bubbles in foaming layers, this layer will be
thicker. The air bubble will take over the place for the upper phase, and the
solution will spill out of the pellet.

6. Sodium acetate: The salts interrupt the hydrogen bonds between the water
and DNA molecules. Sodium ion in sodium acetate and phosphate group of
DNA to form precipitate the DNA

7. In step 8, absolute ethanol helps to remove water from DNA to form shell-
bond precipitate DNA. Trying to make cells out of precipitate DNA.
Moreover, we need to invert slowly. Because the Sodium ion from sodium
acetate will have time to interact with the phosphate group of DNA to
precipitate the DNA.

8. Step 11, incubating in -20-degree Celcius is used for DNA precipitation.

Longer the incubation, qualifier the precipitation.
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GENETICS LABORATORY

Incubate as long as possible (if can: overnight). To reduce the rate of

evaporation. Longer of time, precipitation between the sodium ion with the
phosphate group of DNA will maintain. The efficiency or the use of DNA
extraction is higher. That means all the DNA can be precipitated if the time
is as long as possible.

9. Ethanol 70% used for washing, removing other solute impurities. because
we just need pure DNA. 70% Ethanol has 30% of water which is able to
completely remove supernatant and the absolute Ethanol.

F. Conclusion:

From this experiment, we become aware and understanding of how the

isolation of genomic DNA of plant tissues work