SULTAN QABOOS UNIVERSITY

DEPARTMENT OF CROP SCIENCE

COLLEGE OF AGRICULTURE AND MARINE SCIENCE
TRIBOLIUM CASTANEUM DNA EXTRACTION, PCR
AND GEL ELECTROPHROSIS
Instructor: Dr. Farid Talukder
Abir Omar Hilal AL Rumhi (87142)
Submit on TUE 27/12/2011

INTRODUCTION:
The project deal with one species of insect (T. castaneum) from different place grow in
different condition in order to extract the DNA and distinguish between them. The red
flour beetle is a worldwide stored product pest, flattened, reddish brown color, 3-4 mm
long, 1.2 mm in width. Antennae are bent and bear a distinct club formed by three
enlarged terminal joints. The red flour beetle attacks broken grains, especially wheat
flour. It also feeds upon dry fruits. Both the larvae and adults cause damage to flour, flour
product and grains damaged by other pests and they cause an allergic response but are not
known to spread disease. DNA is the genetic material, the genetic code for life. It is
present in the cells of all living things, including plants and animals, fruits and
vegetables. In eukaryotic the nucleus is protected within a nuclear membrane which is
surrounded by a cell membrane and a cell wall. Four steps are used to remove and purify
the DNA from the rest of the cell. (Lysis, precipitation, Wash and Resuspension).
Polymerase Chain Reaction (PCR), invented by Kary B. Mullis, at the Cetus Corporation,
who was awarded the 1993 Nobel Prize for chemistry for PCR, is a technique to
exponentially amplify in vitro a small quantity of a specific nucleotide sequence in the
presence of template sequence, two oligonucleotide primers that hybridize to opposite
strands and flank the region of interest in the target DNA, a thermostable (taq) DNA
polymerase. The reaction is cycled involving template denaturation, primer annealing,
and the extension of the annealed primers by DNA polymerase until enough copies are
made for further analysis. Gel Electrophoresis is a group of techniques used to separate
molecules based on physical characteristics such as (size, shape and Electrical charge)
there are two types of Gel Electrophoresis Agarose Gel Electrophoresis and Sodium
Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Gel electrophoresis
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used to Separate DNA and RNA molecules by size and Estimate the size of nucleic acid
molecules of unknown length by comparing the total distance traveled by the molecules
to a known value

Figure (1): life stage of tribolium castaneum

Objective:

Provide an overview of the molecular techniques used in insect biotechnology

laboratories

Extraction of genomic DNA from T. castaneum insect

Using PCR program to amplify the genomic DNA with necessary steps

To obtain DNA in a relatively purified form which can be used for further
investigations, i.e. PCR, sequencing, etc

separates molecules on the basis of their rate of movement through a gel under the
influence of an electrical field

using agarose gel electrophoresis to determine the presence and size of PCR
products

Prepare necessary solution for the insect DNA extraction, master mix for
amplification of insect DNA by PCR, agarose gel solution for DNA visualization

Material and Method

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 Insect DNA Extraction

1- Prepare fresh solution A and B in 1.5 ml Eppendorf tube as in the table below

SOLUTIO
N (A)

Reagent(and
strength)
Tris-Base (pH
7.5)(100 Mm)
NaCl (600nM)
EDTA(250mM)
MilliQ water
Total volume:

Amoun
t (l)
20 l

SOLUTIO
N (B)

20 l
40 l
120 l
200 l

Reagent(and
strength)
Tris-Base (pH 9.0)
(3000 Mm)
EDTA(250mM)
Sucrose (20%)
SDS (10%)
MilliQ water
Total volume:

Amount
(l)
20 l
80 l
50 l
25 l
25 l
200 l

2- Place 1-5 live/frozen insect (2 mg) in a 1.5 ml Eppendorf tube, homogenized

using a sterilized micro-pestle

3- Add 100 l of solution A and 100 l of solution B

4- Vortex for 30 seconds, then place the tube in water bath prepared before and
warmed at 60C for 45 min, after that cool the mixture for 15-20 min on the
bench top

5- Add

30 l of 8M potassium acetate to precipitate the protein, and then

incubate for 30 min on ice, after that centrifuge the sample in a microcentrifuge at 14000 xg for 5min at 4 C

6- Carefully

remove the supernatant using micropipette and transfer to a new

Eppendorf and add to it two volume of ice-cold 95% ethanol to precipitate the
DNA

7- Preserve at -20 C for 2 hours, then centrifuge at 14000 xg for 15 min at 4 C,

discard ethanol and add 70% ethanol to wash the DNA pellet, again centrifuge
at 14000 xg for 15 min at 4 C and discard the remaining ethanol

8- Dry the pellet in towel paper and dissolve the pellet in 30-200 l of MilliQ
water depending on the size of DNA pellet, store at -20 C for later use in PCR

Amplification of insect DNA through RAPD-PCR

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 Preparation of PCR sample

9- After thawing all the chemical need in PCR, working insect DNA in step 8
put all in the ice tray

10-

In Eppendorf tube 1.5 ml prepare the master mix following in the table
below:

Master Mix Chemicals Amount

(l)
PCR buffer
dNTP(1Mm)
Primer(10 l)
Taq DNA polymerase(5 U/ l)
MgCl2 (25 mM)
MilliQ water

6 l
6 l
6 l
1.2l
3.6 l
31.2 l

Total:

54l

11- Prepare 0.5 ml PCR tubes and label them (insect and control)
12- Add 18 l master mix for each PCR tubes (insect and control)
13- For insect tube add 2 l working insect DNA and for control tube add 2
l MilliQ water so, thr total volume in each tube = 20 l

PCR analysis
14- Set the PCR machine for an initial denaturation period at 94 C for 1 min one
cycle, followed by 45 cycles as follows:

Steps

Temperature Duration

Cycles

Pre- PCR
denaturation
denaturation
Annealing
Ramp
Extention
Hold temperature

94 C

1 min

94 C
35 C
35 C -72 C
72 C
4 C

1 min
1 min
2 min
2 min

45 cycles
-

15- Put the PCR tubes in the PCR machine, close the led and start the program,
after completion keep the PCR tubes in a refrigerator at 4 C to use it in the
next step Electrophoresis

Electrophoresis and Visualization of insect DNA

refers to the separation of charged particles located in a gel when an electric
current is applied, Charged particles can include DNA, amino acids, polypeptides,
etc witch When the DNA is exposed to an electrical field, the particles migrate
toward the positive electrode, Smaller pieces of DNA can travel further in a given
time than larger pieces, (Electro = flow of electricity, phoresis = to carry across).
A gel is a colloid suspension of tiny particles in a medium, occurring in a solid
form (like gelatin) and Agarose is a polysaccharide made from seaweed.

Figure (2): Red Sea Weed

Preparation of agarose gel:

16- Measure 1.8 g agarose powder in a weighing boat and add it in
a 500 ml reagent bottle
17- Add 100 ml of TBE buffer in the reagent bottle
screw the cap loosely and place the bottle in a microwave oven
for at least 30 seconds
18- If the agarose dissolve completely in the solution take the reagent bottle out
the microwave oven and put it on desktop for cooling at room temperature
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D-galactose

3,6-anhydro L-galactose

Figure (3): Agarose structure

Pouring of agarose gel:

19- Place a gel tray on the table in a horizontal position, add the two gates on the
both ends of the gel tray ,place a comb in the gel tray and pour the warm
agarose gel (50-55 C) into the middle of the gel tray slowly until the agarose
reaches to specific point of comb
20- Remove the comb very slowly when the gel look semi-solid to prevent any
kind of damage, also remove both of gates
21- Fill the gel tank with the proper amount of TBE buffer (provides ions in
solution to ensure electrical conductivity), place gel tray in the tank and check
the level of the buffer (1-2 mm above the gel level)

Preparing PCR product:

22-

Use Pipettor, add 10 l PCR product on waxed side of parafilm paper as a

spot, add 2 l of gel loading buffer (GLB) over PCR product and with the tip
of the pipette mix the mixture very well

23-

Take 10 l mixtures and load it into a well of gel tray. And load 10 l
control PCR product (without insect DNA) in the next well

24-

Load a 5 l of ladder (used as a marker to compare the sample DNA

fragments and estimate their sizes) in the lane (first well) of the gel

Running Electrophoresis:

25-

After PCR product loading, put the electrophoresis lid correctly on the
tank, connect the cables to the power supply unit and turn the switch on after
be sure that the voltage at 105 V

26-

After 1:20 hours, at the end of gel run switch OFF the power unit, remove
the tank lid and gel tray from gel tank

Helpful Hints:

When placing the gel in the electrophoresis chamber:

When adding the buffer to the chamber:

Gently flood the gel from the end opposite the wells to minimize
sample diffusion

Before loading the wells:

Make sure that the wells are closest to the negative (black) electrode
(since DNA is negative)

Orient the entire chamber close to the power supply so it is in place

when the samples are ready to run

When loading samples in the wells of the gel:

Use proper micropipette techniques

Make a written record of which sample you will load in each well of
the gel

Be careful not to puncture the bottoms of the wells as you load the
samples

DNA visualization:
27- Add the processed gel into the EtBr (A fluorescent and mutagen dye
visualized when excited by UV light) tank slowly, close the tank lid and stain
the gel for next 20-30 min

28-

By using spatula or wide spoon after staining, remove the gel from the
EtBr solution and wash it by tap water for 5-10 min to remove the staining
from gel

29-

Place the gel on the Doc system and examine under the UV light

30-

Save the picture of the gel and print a photo

Figure (4): Ethidium Bromide structure

The result:
We did not obtain any band in the gel

Figure (5): picture for the result gel electrophrosis

Discussion and Conclusion:

Since the DNA extract from two different adult T. castaneum the
result should be a mixture of different size of band with different
molecular weight. But the result that obtain were negative, no band
at all in the gel. Many problems can occur during the working lead
to negative results which are Contamination, No or weak product,
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Poor primer, not using "master mixes", wrong staining and Primer
dimers. You can avoid these problems by following these solutions:

Contamination
Reasons are Contaminating DNA can originate from three
sources: DNA from other test samples, DNA from experimental
materials such as recombinant clones, or DNA generated by
previous PCR amplification of the same target sequence. The

solution are Use ultra-clean chemicals, Separate pre- and post

PCR, Always use negative control, Use filtertips , Pipet carefully,
clean gear, Change ingredients, Try somebody elses ingredients,
All surfaces in the PCR area should be routinely decontaminated to prevent cross
contamination use of a DNA decontamination solution, such as dnazap And
wearing gloves

No or weak product
The Reasons are Wrong primers, Missing ingredient, Failed staining, Wrong
conditions, Bad template, Extension time is too short, Cycle number is too low,
dNTPs degraded, Annealing temperature is too high and Problem with
thermocycler operation or program. The Solution are Increase the enzyme
amount, Lengthen the denaturation, Increase the extension time, Increase the
number of cycles, Increase the template amount, Reclean the DNA using ETOH
precipitation, examine template quality via gel electrophoresis, re-prepare
template if necessary, Use fresh dNTPs; store frozen aliquots and avoid freezethaws, Use fresh enzyme, Increase annealing time and Run positive control with
every reaction

Primer dimmers

The Reasons are because of template problems Primers try to anneal to something
and the Solution are to use Positive control, Hot Start, Optimize annealing
temperature and Design new primers which have lower self-annealing or selfhybridization potential
The table below indicates other problem can face during the all procedure

Low
yield
or
no
PCR
produ
ct

2
Nonspecific
PCR
produc
ts

3
Sequen
ce
errors

Low
template
quality or
quantity

Reaction
set-up at
room
temperatu
re

Within a
PCR
product

Faulty
primer
design

Suboptima
l
thermal
cycling
conditions

Suboptim
al
reaction
conditions

Suboptim
al
thermal
cycling
condition
s
Suboptim
al
Reaction
condition
s
Complex
template

Suboptima
l
reaction
conditions

Low
fidelity
polymeras
e

Faulty
primer
design

Sequencin
g
error

4
Product
in
Negative
control

At
PCR
prod
uct
termi
ni
Fault
y
prim
er
desi
gn
Low
prim
er
quali
ty

10
Figur(6
):proble
m of
PCR

Crossover
contamina
tion

RNA
contamina
tion
with
DNA

Abbreviations:
ml

Milliliter

PCR

micrometer

Polymerase chain
reaction
volt

gram

UV

Ultra violet

EtBr

Ethidium
Bromide

Min

Minutes

References:
1- PCR ''Use of PCR '' online www.science director.com accessed 24/12/2011
2- J L Hartley and A Rashtchian contamination in PCR online
www.pubmed.com accessed 22/12/2011
3- Pest control and disinfestations of stored grain pests- manual for btec higher
national diplome in food safety and nygine- compiled by prof. khaja
mohammed azam, sultan Qaboos university, sultanate of oman
4- Lab student protocol (4,5 and 6)
5- Agarose agarose gel electrophrosis online www.science director.com
accessed 24/12/2011

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