MINISTRY OF EDUCATION AND TRAINING

CANTHO UNIVERSITY

BIOTECHNOGY RESEARCH AND DEVELOPMENT INSTITUTE

REPORT
GENOMICS LABORATORY

(BT302C)

SUPERVISOR STUDENTS (Group 8)

PhD. Nguyen Thi Pha Nguyen Nhat Binh B1504424

Vuong Le Thanh Ha B1500894

Tran Huynh Xuan Huong B1504446

Nguyen Pham Thien Trang B1505624

To Huynh Thanh Truc B1504496

CAN THO, 4/2019

 MINISTRY OF EDUCATION AND TRAINING

CANTHO UNIVERSITY

BIOTECHNOGY RESEARCH AND DEVELOPMENT INSTITUTE

REPORT
GENOMICS LABORATORY

(BT302C)

SUPERVISOR STUDENTS (Group 8)

PhD. Nguyen Thi Pha Nguyen Nhat Binh B1504424

Vuong Le Thanh Ha B1500894

Tran Huynh Xuan Huong B1504446

Nguyen Pham Thien Trang B1505624

To Huynh Thanh Truc B1504496

CAN THO, 4/2019

i
 INDEX

INDEX .................................................................................................................................. i
LIST OF TABLES ..............................................................................................................ii
LIST OF FIGURES ............................................................................................................. i
LESSON 1: DNA EXTRACTION .................................................................................... 1
1.1 MATERIALS ........................................................................................................ 1
1.2 PROTOCOL .......................................................................................................... 2
LESSON 2: TESTING DNA BY GEL ELECTROPHORESIS ..................................... 4
2.1 MATERIALS ........................................................................................................ 4
2.2 PROTOCOL .......................................................................................................... 4
2.2.1 Preparation of agarose gel .............................................................................. 4
2.2.2 Running of electrophoresis .............................................................................. 4
2.3 RESULTS AND DISCUSSION ............................................................................... 6
LESSON 3: GENETIC DIVERSITY ANALYSIS OF RICE USING RAPD
TECHNIQUE ...................................................................................................................... 8
3.1 MATERIALS ........................................................................................................ 8
3.2 PROTOCOL .......................................................................................................... 8
3.2.1 RAPD PCR ....................................................................................................... 8
3.2.2 Agarose electrophoresis ................................................................................... 9
3.3 RESULTS & DISCUSSION............................................................................... 11
LESSON 4: GENETIC DIVERSITY ANALYSIS OF RICE BY USING SSR
(SIMPLE SEQUENCE REPEATS) TECHNIQUE ...................................................... 12
4.1 OBJECTIVE ....................................................................................................... 12
4.2 MATERIALS AND CHEMICALS ................................................................... 12
4.3 PROTOCOL ........................................................................................................ 12
4.4 RESULTS AND DISCUSSION ......................................................................... 14

ii
 LIST OF TABLES

Table 3. 1: Chemical substances mixture for RAPD technique (1 reaction) .............. 10

Table 4. 1: Chemical substances mixture for SSR technique (1 reaction) .................. 12

iii
 LIST OF FIGURES

Figure 2. 1. DNA bands after analyze with Gel reader Bio-Rad machine .................... 6

Figure 3. 1. Thermal cycle for PCR-RAPD .................................................................... 10

Figure 3. 2. Result of PCR-RAPD using OPG-04 primer ............................................. 11

Figure 4. 1. Thermal cycle for PCR-SSR ....................................................................... 13

Figure 4. 2. Result of SSR-PCR using RM7075 primer ................................................ 14

iv
 LESSON 1: DNA EXTRACTION\

DNA technique involves admitting the total amount of DNA with high purity,
content enough, less fracture to serve for the study of organism genetics. DNA is the first
important material for the genome studies as well as the gene research, molecular biology
genetic materials such as PCR, SSR, RAPD, AFLP, RFLP, ...

DNA is a genetic material in the nucleus cell protecting by proteins, cell membrane
system, especially plant cells also have cellulose membranes protected. The general
principle to obtaining the complete and intact DNA and how to remove DNA from this
protection system. Mechanical effects such as grinding samples in cold conditions, or in
appropriate buffers with the involvement of certain chemicals, will help release DNA into
the extract and DNA is recovered by the role of ethanol. Depending on the subject of
DNA extraction, the chemicals used in each process may be different.

1.1 MATERIALS
- Leaves from plant in dish 5 (Particularly rice- Oryzae sativa 2n=24, OM4900 for
group 8)
- Mortar, cotton balls, scissors (each group use different scissor in order to avoid
DNA confusion), clamps, type 2mL eppendorf, foil, marker, etc.
- Micropipettes 2-20µL, 10-100µL, 100-1000µL
- Water bath 65oC
- Centrifuges, moist heat sterilization pot, vacuum dryers, refrigerators -20oC
- EB buffer: 0.1M Tris HCl (pH 8), 0.5M NaCl, 0.05 EDTA, 0.01 -mercaptho
ethanol
- TE buffer : 10mM Tris pH 8, 0.1mM EDTA (pH 8)
- CTAB buffer : 0.2M Tris.HCl (pH 7,5), 2M NaCl, 0.05M EDTA, 2% (w/v) CTAB
(cetyltrimethylammonium bromide)
- SDS 10%
- Mixed Chloroform- Isoamyl alcohol 24:1
- Isopropanol
1
- Ethanol 96%
- Ethanol 70%
 The role various components of DNA extraction protocol is as follows:
- Cetyl trimethyl ammonium bromide (CTAB) or SDS => disrupts the membranes.
Moreover, DNA and protein have different solubilities in CTAB. Protein are
insoluble while DNA is soluble.
- β mercaptoethanol => helps in denaturing proteins by breaking the disulfide bonds.
- EDTA => chelates the magnesium ions required for DNase activity.
- Buffer is almost always Tris at pH 8 and a salt such as sodium chloride => aids in
precipitation by neutralizing the negative charges on the DNA so that the
molecules can come together.
- TE buffer => protect DNA from degradation.
- Mixed Chloroform- Isoamyl alcohol 24:1 => precipitate all undesirable
contaminants that are chiefly made of proteins so that nucleic acid solution is
extracted successively.
- Alcohol => for nucleic acid precipitation and washing.

1.2 PROTOCOL
- Washing leaf with water and subsequently cleaning by alcohol
- Grounding the samples with 1mL EB solution
- Taking out 1mL extracted solution and then transfer to tube 2.2mL
- Adding 50µL SDS 10% and mix well
- Incubating at 65oC in 30 minutes
- Centrifuging at 13000 rpm in 10 minutes
- Taking out 800µL upper solution, transferring to a new tube and adding 800µL
isopropanol, shaking tube slightly
- Incubating at -20oC in 1 week, store for the next steps
- Centrifuging at 13000 rpm in 10 minutes. Keeping precipitated part at the
bottom of the tube, removing the solution

2
- Adding to tube 400µL TE 1X (pH 8), 400µL CTAB buffer and incubating at
65oC in 15 minutes
- Adding 800µL Chloroform: Isoamyl alcohol solution at the ratio of 24:1 and
shake well
- Centrifuging at 12000 rmp in 5 minutes
- Transferring carefully 600 µL solution at phase 1 to another tube, adding 1.2
mL ethanol 96% and incubating at room temperature in 15 minutes
- Centrifuging at 13000 rpm in 10 minutes, getting rid of the solution and
keeping
- Drying tube with DNA by using vacuum at 45oC in 10 minutes
- Dissolving in 80µL BiH2O to store DNA.

3
 LESSON 2: TESTING DNA BY GEL ELECTROPHORESIS
2.1 MATERIALS
- DNA samples (DNA samples were extracted in lesson 1)
- Agarose
- Electrophoresis buffer (1X TBE buffer)
- Loading buffer
- Electrophoresis kit
- Micropipettes, pipette tips
- Parafilm paper
- Microwave (using at the preparation step of agarose gel)
- Conical flask (using at the preparation step of agarose gel)
- Documentation system (Gel Reader Bio-Rad machine)

2.2 PROTOCOL

2.2.1 Preparation of agarose gel

- Prepare 1X TBE buffer

- Transfer 100mL of the buffer to conical flask
- Weight 1 gram of agarose and add to the 100mL buffer solution
- Heat solution flask in microwave oven until completely dissolved
- Cool the solution to about 60℃, add safe view
- Pour the solution to a gel caster tray for solidification
- Place the comb, after the gel solidification, the comb is removed.

2.2.2 Running of electrophoresis

- Sample containing DNA (15µl) mixed with 3µl loading buffer by micropipette
and then pipetted into the sample wells.
- Connect the electrodes and switch on the current. Apply a voltage of 100V.
Run the gel until the loading buffer dyes have moved ab appropriate distance
through the gel.

4
- The gel tray may be removed and placed directly on the gel documentation
system to image and save.

5
2.3 RESULTS AND DISCUSSION

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Gel 1 (15 wells)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Gel 2 (17 wells)

1 2 3 4 5 6

Gel 36 (6 wells)
Figure 2. 1. DNA bands after analyze with Gel reader Bio-Rad machine
Results analysis:
After running gel electrophoresis:
- There were 25 samples have the results that indicated DNA band: well number
6, 7, 9, 11, 12, 13, 14 (gel 1); well number 1, 2, 3, 4, 5, 10, 11, 12, 13, 14, 15,
16, 17 (gel 2); well number 1, 2, 3, 4, 5, 6 (gel 3).
- Other wells have no showed DNA band: well 1, 2, 3, 4, 5, 15 (gel 1); well 6, 7,
8, 9 (gel 2). Due to the process of cell disruption, it is impossible to break down
since DNA extraction step (extracting skills are not good). Therefore, there is
no DNA.
- Wells have a large light on the gel: well number 6, 9, 10 (gel 1); well number 1,
2, 3, 10 (gel 2); well number 5, 6 (gel 3). Due to the DNA samples were
contaminated.

7
LESSON 3: GENETIC DIVERSITY ANALYSIS OF RICE USING RAPD
TECHNIQUE
3.1 MATERIALS
- DNA template from lession 1.

- RAPD primer: OPF 08

- Chemicals: Bi.H2O, Buffer 5X and dNTPs, Taq polymerase, Tris-HCl (pH 8),
loading buffer 6X for electrophoresis, TBE 1X, EDTA, agarose, ladder.

- Tools: Micropipette (20µl and 200µl), Eppendorf tubes 1.5mL and 0.2mL.

- Machine: PCR machine, Gel-reader Bio-rad machine, Electrophoresis

Equipment minisub gel.

3.2 PROTOCOL

3.2.1 RAPD PCR

- Prepare one 1.5mL Eppendorf tube to contain the mixture for RAPD excepting
DNA template and four 0.2mL Eppendorf tubes (PCR tubes) for carrying out
PCR.

- Use micropipette to drag chemicals showed in table 3.1 into 1.5mL prepared
eppendorf respectively excepting DNA template.

Note:

 The volumes of the substances have to be multiplied by 5 before adding

to 1.5mL Eppendorf tube to make sure that there is enough mixture for 4
reactions.

 Taq polymerase is very sensitive with temperature, so after adding 3 first

components, move this tube to the refrigerator and add Taq polymerase.

- Mix the tube well and separate the mixture into 5 prepared 0.2mL Eppendorf
tubes with 13µl of volume for each tube.

8
 - Add 2µl DNA template in these 0.2mL Eppendorf tubes.

+ Tubes number 1, 2, 3 and 4 contain DNA from lesson 1.

+ Tube number 5 contains sample DNA in lab.

Each PCR tube contains 15µl mixture in total.

- Set program for PCR machine as in figure 3.1 and start the machine. In this
experiment, the cycles in PCR was increased to 45 cycles to reduce the effect of
reproducibility of RAPD on result.

3.2.2 Agarose electrophoresis

- Prepare 1.5% agarose gel.

- Prepare a piece of parafilm to carry droplets of loading buffer, each droplets is

2µl.

- Use micropipette to drag and mix well 15µl PCR product with droplets of
loading buffer on parafilm. Then put all the mixture on the wells in gel.

- Pour TBE 1X into electrophoresis box after putting the gel on.

- Inject ladder solution on the 1st well.

- Connect the electrodes, operate the machine with 50V electric current and
switch on the current.

Note:

 Pay attention to the tracks of bands and do not let the color stain track
run out of the gel. The proper point to stop electrophoresis is when the
stain color runs 2 out 3 the height of gel.

- Remove the gel tray and place the gel directly on the Gel-reader machine to
image and save.

9
Table 3. 1: Chemical substances mixture for RAPD technique (1 reaction)

Chemical substances Volume (µl)

Bi.H2O 8.65

Buffer 5X 3

Primer RAPD 1.2

Taq polymerase 0.15

DNA 2

Total volume 15

After mixing up those compositions as table 1 (the number of reaction carry

out by each group). To separate the mixture into tubes 0.2ml in such a way that
each tube contains 13 µl, then add 2µl DNA sample to reach 15µl
Set program for PCR machine as the following thermal cycles:

94oC 94oC
72oC 72oC
5:00 0:15
1:20 10:00

35oC
0:15
10oC
∞
45 cycles ∞

Figure 3. 1. Thermal cycle for PCR-RAPD

10
3.3 RESULTS & DISCUSSION

Ladder 14 15 16

Figure 3. 2. Result of PCR-RAPD using OPG-04 primer

(Well 14: Rice 1, well 15: Rice 3, well 16: lab rice)

According to figure 3.2, the result of RAPD is rather good. The brightest in well 14
and 15 are similar size (~500bp compare to ladder) that demonstrate 2 rice strands are
monomorphic. Cause may be 2 rice samples have closely family. In well 16, lab
sample is too little to extract enough the amount of DNA (2l) or the lid tube was not
tight and had spilled sign. It can be reason we can’t see any bands on gel. We limited
the low reproducibility of RAPD marker by PCR cycle increase to the primer catch as
many position as possible. Therefore, the result almost give full number of bands. In
addition, from this results, it is possible to assert that RAPD technique cannot
distinguish among genetically close strains because all the rice strains used in this
experiment are salt-tolerant strains having genetically close relations.

11
 LESSON 4: GENETIC DIVERSITY ANALYSIS OF RICE BY USING SSR
(SIMPLE SEQUENCE REPEATS) TECHNIQUE

4.1 OBJECTIVE
Application for selecting the parents to create the new hybrid lines with high quality.

4.2 MATERIALS AND CHEMICALS

- DNA template (from lesson 1)
- Tools and chemical substances: Micropipette, electrophoresis equipment
minnisub gel, gel reader Bio-rad machine, eppendorf tubes 1.5mL and 0.2mL,
PCR machine, agarose, loading buffer 6X, SSR primers RM7075
Primer RM7075 was used in experiment. These are information about primer:
- Marker ID: 2499055
- Species: Oryza sativa Japonica Group
- Sequence source: AY02375
- Primer forward: 5' TATGGACTGGAGCAAACCTC 3'
- Primer reverse: 5' GGCACAGCACCAATGTCTC 3'
- Repeat motif: (ACAT)13
- Anneal temperature: 50
- Product size: 155pb

4.3 PROTOCOL
- Prepare 1.5mL eppendorf to contain the mixture which in turn will be
separated to many tubes for PCR to be carried out. In high concentration,
chemical reactions will occur easily between substances so it should prepare
Bi.H2O before adding other subtances.
- Use micropipette to drag those chemical subtances in the following table into
1.5mL eppendorf respectively.

Table 4. 1: Chemical substances mixture for SSR technique (1 reaction)

12
 Chemical subtances Volume (µL)

Bi.H2O 8.65

Buffer 5X 3

Primer RM7075 F 0.6

Primer RM7075 R 0.6

Taq polymerase 0.15

DNA 2

Total volume 15

- After mixing up those compositions as table 4.1 (the number of reaction carry
out by each group). To separate the mixture into 0.2 mL tubes in such away
that each tube contains 14 µL, then add 1 µL DNA sample to reach 15 µL.
- Set program for PCR machine as in Figure 4.1 and start the machine.

Figure 4. 1. Thermal cycle for PCR-SSR

13
4.4 RESULTS AND DISCUSSION

- After got the PCR products, electrophoresis was carried out and the results was
got from 39th to 42nd wells (Figure 4.2).

Figure 4. 2. Result of SSR-PCR using RM7075 primer

(Well 39: Rice 1, well 40: Rice 3, well 41: Rice 2, well 42 lab sample )

- In well 42, lab sample is too little to extract enough the amount of DNA (2l) or
the lid tube was not tight and had spilled sign. It can be reason we can not see
any bands on gel.

14