Polymerase Chain Reaction and HPLC 1 PCR and HPLC 2 1

PCR and HPLC 1
PCR AND HPLC
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PCR and HPLC 2
1. Practical Report: Polymerase Chain Reaction (PCR)
Introduction
PCR is a technique applied in molecular biology to amplify an individual copy or
substantial copies of DNA. PCR involves 20-40 cycles of temperature changes (Bartholomew et
al., 2016). The process has six steps including initialisation, denaturation, annealing, extension,
final elongation, and final hold. Denaturation step consists in increasing temperatures to separate
the strands in the double helix. During the annealing process, the temperature is lowered to allow
the binding of the primer to the single strands that should undergo amplification. The extension
step involves the introduction of Taq polymerase that catalyses the formation of complementary
strands from the separated helix. This report will discuss the application of PCR in the process of
DNA amplification.
The aim of the Experiment
The objective of the experiment is to amplify (reproduce) the specified segments of DNA
using Polymerase Chain Reactions. The amplicons are useful in several molecular applications
like gel electrophoresis among others.
Materials and Methods
Materials
The materials include primers, DNA polymerase (Taq), DNA template, dNTPs, sterile
water, and magnesium salt (Jalali, Zaborowska, and Jalali, 2017). The primers should be
appropriate to the target DNA. The buffer should also be specific to the Taq polymerase. Other
materials include micropipettes, ethanol resistant marker, thermal cycler, PCR caps, tubes, and
tube rack.
Method

PCR and HPLC 3
Place the well plate into an ice bucket to hold the PCR tubes. The cold conditions prevent
nonspecific priming and nuclease activity (Tung et al., 2015). Pipette the PCR reagents into the
PCR tube which is inside the ice bucket. Add the reagents starting with sterile water, then buffer,
followed by dNTPs, Magnesium salt, primers, and lastly template DNA. Add every reagent in
PCR tube except the DNA template to form the negative control. Close the PCR tubes using their
cups and put them inside the thermal cycler. Start the program after ensuring that the thermal
cycler lid is closed. At the end of the experiment, remove the PCR tubes and store them at 4ºC.
Results
Interpreting the results of the experiment involves loading aliquots of the reactions into
agarose gel wells and staining DNA using ethidium bromide (Hazzalin, and Mahadevan, 2017).
The staining permits the visualisation of amplified DNA through the UV illuminator. The PCR
experiment worked well since the separation of DNA was evident during electrophoresis.
However, the blanks can be due to contamination of DNA or nonspecific amplification.
Discussion
Gel electrophoresis shows whether the experiment was a success or otherwise. The
results indicate successful amplification due to correct adherence to the PCR procedures
(Hazzalin, and Mahadevan, 2017). Contamination of DNA with molecular substances like
proteins, RNA prevents amplification. Primer dimerisation also prevents successful
amplification.
Conclusion
PCR assists in the amplification of DNA strands, and it has six steps. The required
materials for the experiment include a polymerase, sterile water, primers, dNTPs, and
magnesium salt among others. Visualisation of the amplification process involves running the

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products of PCR into gel electrophoresis. Accurate adherence to the procedure yields desirable
results; however, contamination and primer dimerisation can prevent amplification.