AIMST UNIVERSITY

FACULTY OF APPLIED SCIENCES

LEARNING OUTCOMES EVALUATION FORM

COURSE TITLE / CODE : SCBT 33114 BIOMOLECULAR DIAGNOSTICS

NAME: LIM WEI YANG

MATRIC NO.: S18101039 BATCH: 42

Please tick Lab Report Assignment Project Practical No.:

√
√ 3
TITLE: Real-Time Polymerase Chain Reaction

DATE OF SUBMISSION: 22th JUNE 2021

Descriptors Marks Evaluated

(max 20) Marks
Not able to organizes and perform experiments even with constant supervision 0
Not able to use the light microscope and other equipment
Shows no understanding and engagement in conducting experiments
Not able to generate any data for scientific report
Organizes and performs experiments with limited success even with constant supervision 1-5
Uses the light microscope and other equipment with poor precision, accuracy and skill
Shows poor understanding and engagement in conducting experiments
Generates inaccurate and unreliable data for scientific report
Organizes and performs experiments safely and successfully with constant supervision 6-10
Uses the light microscope and other equipment with minimal precision, accuracy and skill
Shows limited understanding and engagement in conducting experiments
Generates partially accurate and reliable data for scientific report with assistance
Organizes and performs experiments safely and successfully with minimal supervision 11-15
Uses the light microscope and other equipment with some precision, accuracy and skill
Shows good understanding and some engagement in conducting experiments
Generates accurate and reliable data for scientific report with assistance
Organizes and performs experiments safely and successfully without supervision 16-20
Uses the light microscope and other equipment with good precision, accuracy and skill
Shows full understanding and engagement in conducting experiments
Generates accurate and reliable data for scientific report independently

Comments: ______________________________________________________________________
________________________________________________________________________________

Signature of Lecturer : _____________________ Date : ___________________

Practical 03: Real-Time Polymerase Chain Reaction

PO4 - Ability to analyse, synthesise and integrate knowledge and information

1. Comment on the curve pattern of the amplification plot for the following samples:
i. negative control

The curve pattern of amplification plot for negative control is parallel with no
amplification occurs. If there is amplification occurs, indicating that there are
contamination of the reagent. Thus, from the result shown, negative control
starting to amplify after 28 cycles of PCR. Hence, the low-level signal which is
below the threshold indicates there is a background noise.

ii. serial dilutions of gDNA

The curve of serial dilutions of gDNA shown are exceed the threshold line. The
Relative Fluorescent Unit (RFU) of serial dilutions of gDNA are about 490.00,
490.00, 440.00, 400.00, and 260.00, respectively. Additionally, the first amplicon
crosses the threshold after 12 cycles of PCR. The second after 15 cycles. The third
after 19 cycles. The fourth after 24 cycles. The last after 26 PCR cycles.

iii. test sample

The curve of test sample is exceed the threshold line after 20 PCR cycles. The
Relative fluorescent Unit (RFU) of test sample shown is about 500.00.

2. Comment on the standard curve and the correlation coefficient value obtained.

The slope of the standard curve obtained is -3.523 which represents a good reaction as it
is between slope -3.58 and -3.10 as well as efficiency between 90% and 110%. The
correlation coefficient is a measurement of the performance of reaction, reflecting the
linearity of the standard curve. The correlation coefficient value obtained is 0.9983 which
is an ideal value as it is closer to 1.

3. Using the standard curve, estimate the starting concentration of gDNA in the test sample

When Ct value of test sample is21 ,

Y =−3.5225 x +16.231
21=−3.5225 x +16.231
21−16.231=−3.5225 x
−3.5225 x=4.769
X =4.769 /(−3.5225)
¿ log 1.354(ignore the negative sign)
The log startingconcentration is1.354 ng .
X =log 1.354
= 0.1316
 ¿ 0.132 ng/ μl
Therefore, the starting concentration of gDNA in the test sample is 0.132 ng/μl

4. How many peaks were observed in the melting curve analysis? Which of the peaks
correspond to the PCR amplicon?

One peak is observed in the melting curve analysis. The test sample peak is
corresponding to the PCR amplicon.

PO1: Ability to demonstrate a comprehensive understanding of Biotechnology

5. At which phase of the PCR amplification is the fluorescence signal measured?

Exponential phase.

6. Why is it better to measure the fluorescence signal at this phase?

Exponential phase shows the doubling of amplicon occurs every cycle. During
exponential phase, the amount of PCR product present in the reaction tube is directly
proportional to the amount of emitted fluorescence and the amount of the initial target
sequence. Hence, it truly represent the initial amounts of starting target material. The
reaction is said to be very specific and precise.

7. Besides SYBR Green, name 2 other real-time PCR platforms.

The 5’ nuclease assay (Taqman) and Molecular Beacons

8. List 2 advantages of real-time PCR compared to agarose gel electrophoresis.

The real-time PCR measures the amount of the product during exponential phase while
the PCR with agarose gel electrophoresis measures the amount of the product during
plateau phase. Besides that, PCR with agarose gel electrophoresis is time consuming and
non-automated compared with real-time PCR. Furthermore, Real-Time PCR is designed
to collect data as the reaction is proceeding, which is more accurate for DNA and RNA
quantitation and does not require laborious post PCR methods. Additionally, detection of
Real-Time PCR is capable down to a 2-fold change is also one pf the advantages.

9. State a precaution that should be practiced when performing real-time PCR.

Gloves must be always worn during the experiment. This is because the real-time PCR is
sensitive to the contamination. The contamination of the sample will cause the reading to
become incorrect. The pipette tip should always change while handling different samples
to prevent cross contamination. Furthermore, regularly decontaminate surfaces and
equipment that are utilized for preparing qPCR reactions is also a very important
precaution step.