Polymerase chain reaction (PCR) is a molecular technique used to amplify specific regions of

DNA for applications such as sequencing and genetic analysis. Typically, there is a limited
amount of DNA in the sample to study and amplification is required. PCR is carried out in a test
with a DNA template, primers specific for the region that is desired, DNA polymerase, and
reagents that stabilize the reaction. Once the reaction is put together, it will go into a
thermocycler (PCR machine) that will create the conditions for DNA replication to occur. PCR
required three stages which are the denaturation stage, annealing stage, and elongation stage
(PCR and Gel Electrophoresis, 2021).

There are certain essential components and reagents in PCR, as well as three steps of the PCR
cycle that complete this experiment. The master mix solution in this experiment must be
prepared with PCR buffer (TE buffer), which functions to stabilize the DNA polymerase and
maintain the appropriate pH of the PCR reaction. Following that, the master mix solution
requires dNTPs, which provide energy and the building blocks nucleotides for the synthesis of
new DNA strands, MgCl2 used in PCR reactions for Taq DNA polymerase activity, and forward
and reverse primers to bind towards the target sequence for complementary a template DNA
sequence. Following that, the PCR cycles are completed by denaturation, annealing, and
elongation. Denaturation is the process that occurs when the DNA template denatures due to the
breakdown of hydrogen bonds between complementary bases. The Annealing stage then allows
the primers to be annealed to the single-stranded DNA template. The annealing temperature must
be low enough to allow primer-strand hybridization yet high enough to allow selective
hybridization. Finally, the thermophilic polymerase can add complementary nucleotides to the 3'
end of the primers during the Elongation stage.

When doing a laboratory experiment, precautions must be taken carefully to avoid any errors or
the experiment would fail. First, it's essential to be ready while starting a PCR experiment. Wear
gloves to prevent contaminating the reagents or reaction mixture (Lorenz, 2012). Next, avoid
using a tip that has previously been used to take another reagent since this could result in cross-
contamination because the tip will come into touch with the other reagent (Bryan, 2022).
Moreover, in order to maintain the integrity of the samples and reagents and prevent heat
degradation, PCR reagents must always be kept on ice (Why are the PCR reagents stored on the
ice at all times?, 2022). Last but not least, be careful to select the proper annealing temperature.
When a particular primer set is annealed at the incorrect temperature, errors might be seen in the
results of PCR (Goldberg, 2020).
As shown in the table, our findings stated that the volume for 1x µl can be obtained using the
formula M1V1 = M2V2 where M1 represents the value for working concentration, M2 represents
the final value, V2 is the total volume which is 25 µl and V 1 is the 1x µl volume. The master mix
solution can be obtained by mixing the dNTPs, PCR buffer, MgCl 2, forward primer, and reverse
primer. The temperature annealing for forward primer and reverse primer can be obtained by
using formula 4(GC) + 2 (AT) and the annealing temperature for the experiment is obtained
using Ta = (Taf + Tar / 2) – 5 which results in 53°C. While the calculation is pretty
straightforward, it can lead to complications of the result if wrong calculations or volumes are
being used.

LibreTexts Biology (2021). PCR and gel electrophoresis. Retrieved from

https://bio.libretexts.org/Courses/North_Carolina_State_University/
MB352_General_Microbiology_Laboratory_2021_(Lee)/08%3A_Bacterial_Identification/
8.05%3A_Lab_Procedures-_PCR_and_Gel_Electrophoresis

References

Bryan, B. (2022). The do’s and don’ts of re-using pipette tips. Retrieved from mini pcr bio:
https://www.minipcr.com/re-using-pipette-tips/

Goldberg, A. (2020). 13 Easy Tips for Avoiding PCR Errors. Retrieved from LABTAG:
https://blog.labtag.com/13-easy-tips-for-avoiding-pcr-errors/

Lorenz, T. C. (2012). Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and
Optimization Strategies. Retrieved from PubMed Central:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846334/#:~:text=Wear%20gloves
%20to%20avoid%20contaminating,a%20reaction%20(Figure%202).

Why are the PCR reagents stored on ice at all times? (2022). Retrieved from AAT Bioquest:
https://www.aatbio.com/resources/faq-frequently-asked-questions/Why-are-the-PCR-
reagents-stored-on-ice-at-all-times