Lab Report BET305 - Rahmah Hayati Binti Mohd Fauzi
Lab Report BET305 - Rahmah Hayati Binti Mohd Fauzi
Lab Report BET305 - Rahmah Hayati Binti Mohd Fauzi
LAB REPORT
INSECT MOLECULAR GENETICS
SEMESTER 1 2020/2021
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Insect molecular genetics is the study of molecular structure of DNA in insect that
systematics and ecology. In this study, understanding the routine procedure in the
laboratory is fundamental to get the information of the desired DNA. Method such as
Polymerase chain reaction (PCR), DNA extraction, Gel Electrophoresis and The
Northern blot are commonly used in laboratory. Gel electrophoresis is the technique that
and sibling species (Avise, 1974). In this practical, we require to practice technique to
lyse the cells gently and solubilise DNA. Besides, removing contaminating protein, RNA
Coptotermes gestroi. The result of DNA fragments extracted is used to determine DNA
by a large, mature colony has resulted in a very bad condition to the wooden structure
(Scheffrahn, R. H., & Su, N. Y., 2000). This species has been found in many parts of the
world such in Asia, New World tropics (Brazil and Barbados) southern Mexico, the
Southeastern United States, some islands of the West Indies, the Marquesas Islands
(Pacific Ocean), Mauritius and Reunion Islands (Indian Ocean) and recently in Taiwan
A) DNA Extraction
Five soldiers of termites of Coptotermes gestroi is washed with 70% ethanol then
rinsed with ddH2O. The head of the termites is cut by using forceps and transferred into
1.5 ml microcentrifuge tube. 200 μl of QGT buffer is added and the sample is
added and shook vigorously for 10 seconds. 200 μl of absolute ethanol is added to
sample lysate and is mixed immediately by shaking vigorously for 10 seconds. The GD
14000rpm for 30 seconds then the flow-through is discarded. The GD Column is placed
back into the Collection Tube. 600 μl of Wash Buffer is added to the GD Column. The
Next, the sample is centrifuged for 3 minutes at 14000rpm to dry the column matrix.
The following steps are repeated twice. The GD Column that is dried being transferred
the center of the column matrix. The sample is stood for at least 3 minutes to allow the
Components Amount ( μl ¿
Template DNA 5
Total volume 25
2. The mixture is resuspended gently. The PCR tube is placed into a thermal cycler.
Pre-denaturation 95.0 5 1
Denaturation 95.0 1
Annealing 55.0 1 35
Extension 72.0 2
Store 4.0 ∞
3. After completing the cycles, the PCR tube is removed from the thermal cycler
C) Gel Electrophoresis
until agar dissolved. The liquid is let to slightly cooled down by water bath and mixed
with 5 μl volume of RedSafe nucleic acid stain. The buffer is poured into gel cast and
the gel comb is put on. The buffer is left to solidify in room temperature for 20-30
minutes. The gel comb is removed. The gel cast is transferred to gel tank and is
sample (DNA extraction and PCR reaction) is mixed with the DNA loading dye by
pipetting. The DNA mix is pipetted into the consecutive well. 5 μl of thermo Scientific
GeneRuler 1 kb DNA ladder is pipetted into the first well. Gel electrophoresis is
1. Explain in detail the mechanism of gel electrophoresis and the use of Agarose in
molecular genetics. Agarose has been widely used in DNA separation as it is the most
effective way to separate DNA fragments of varying sizes ranging from 100 bp to 25
kb1. Agarose polymers binds non-covalently and form a network of bundles whose
pores sizes determine a gel’s molecular sieving properties. The sizes of DNA fragments
that will be separated will determine the concentration of the agarose in a gel. Most of
As the DNA that mixed with loading dye is loaded into pre-cast well in the gel, the
current is applied. The DNA fragments move to the positive electrode because the
phosphate backbone of the DNA molecule is negatively charged. DNA has a uniform
mass or charged ration thus is easier to separate by size in a pattern that shows the
distance travelled is inversely proportional to the log of its molecular weight. The ladder
that we observed under UV light is the separated DNA that determine the sizes of the
bands. The sizes of DNA fragments can be determined by comparing to the DNA
standard or DNA ladders. (Lee, P. Y., Costumbrado, J., Hsu, C. Y., & Kim, Y. H., 2012)
2. Describe what you observed on the gel (include details such as smear, bands at
what size etc.) and explain your observation or reasons to the observation.
From the gel electrophoresis, our group (Group 6) has obtained one band of DNA
fragments. By comparing with the Thermo Scientific GeneRuler 1kb DNA ladder in the
lane 1, we approximate the band is 10 000 bp in size. However, the band is slightly
blurred. This might the result of contamination during the procedure of extracting DNA.
The Pipette tip that has been used might contaminated with bacteria or chemical. The
pipette tip should have been directly immersed first in the chemical without touching any
surface. Some of the chemical used such as ethanol is also contaminated during the
preparation. Apart from that, another possible error might arise from the well of the gel
electrophoresis. The comb of the electrophoresis might be inserted wrongly or the gel is
disturbed during the removal of the comb. As a result, the DNA sample unable to come
3. Briefly describe what DNA ladders are and how they were generated.
DNA ladders are common reagent composed of known DNA sizes that used to
determine the size of the sample DNA fragment. The sample DNA fragment will be
separated by electrophoresis along with the DNA ladder according to the band of known
sizes.
For example, if the insertion has ten tandem repeats of a 100 bp, we will obtain 100
bp DNA ladder with fragments from 100 bp to 1000 bp. Cloning, PCR and Partial
restriction with restriction enzyme are used to produce DNA ladder. DNA fragments of
100 bp with unique restriction site at both ends are self-ligated to create a tandem
repeat. Once being cloned, the tandem repeat was rapidly amplified by PCR. Tandem
repeat of a DNA fragment is inserted into a plasmid. In each junction of repeat units,
there are the same unique restriction site. A partial restriction digestion of this plasmid
produces a ladder containing multimers of the repeated DNA fragment (Lan, V. T. T.,
Loan, P. T. T., Duong, P. A. T., Thanh, L. T., Ha, N. T., & Thuan, T. B., 2012).
bromide.
To visualise the DNA fragments in agarose gel, the ethidium bromide is usually used
as dye. Under UV light, the excitation and fluorescence emitted by EtBr at maximum
500- 590 nm. This protocol should be performed rapidly to prevent the DNA to degrade
by the long exposure of the UV light. Apart from it, EtBr also a hazardous waste and
After the exposing in the UV light, electrons in the aromatic ring of the ethidium
molecule are activated. The light energy is released as the electrons return to the
ground state. EtBr is an intercalating agent which resembles a DNA base pair. EtBr
works by intercalating itself in the DNA molecule depending with concentration of the
EtBr. Thus, the fluoresces emitted by EtBr within DNA molecule. In addition, EtBr
re-emits this energy as yellow or orange light with 560 nm absorbance. We are allowed
to observe the DNA fragments based on the intensity. Because of the positive charged,
DNA from the soldier termites of Coptotermes gestroi. To get the DNA, the head
area of the Coptotermes gestroi is cut as it is easier to breakdown the cell because
the exoskeleton is softer compared to other regions. If other than head region is
used, we might not see the band in the gel electrophoresis. The result of the gel
extraction. We should redo the procedure and practice precaution to get the best
result.
References
Avise, J. C. (1974). Systematic value of electrophoretic data. Systematic Biology, 23(4), 465-481.
Lan, V. T. T., Loan, P. T. T., Duong, P. A. T., Thanh, L. T., Ha, N. T., & Thuan, T. B. (2012). Straightforward
procedure for laboratory production of DNA ladder. Journal of nucleic acids.
Lee, P. Y., Costumbrado, J., Hsu, C. Y., & Kim, Y. H. (2012). Agarose gel electrophoresis for the separation
of DNA fragments. JoVE (Journal of Visualized Experiments), (62), e3923.
Scheffrahn, R. H., & Su, N. Y. (2000). Asian subterranean termite, Coptotermes gestroi (= havilandi)
(Wasmann)(Insecta: Isoptera: Rhinotermitidae). Extension document EENY-128. Institute of Food
and Agricultural Sciences, University of Florida, Gainesville, FL.
Yeap, B. K., Othman, A. S., & Lee, C. Y. (2011). Genetic analysis of population structure of Coptotermes
gestroi (Isoptera: Rhinotermitidae) in native and introduced populations. Environmental
entomology, 40(2), 470-476.