Experiment Title : POLYMERASE CHAIN REACTION (PCR) &

ANALYSIS OF PCR PRODUCT

Subject : UDEE2124 PRINCIPLES OF BIOTECHNOLOGY

Course : BACHELOR OF SCIENCE (HONS) BIOCHEMISTRY

Name of Student AHMAD FAIZUL 1405439

:

Date of : 14/ MARCH/ 2017 & 21/ MARCH/ 2017

Experiment

Name of Lecturer : MR. KAM YEW CHEE

Experiment 4

TITLE: Polymerase Chain Reaction (PCR) and analysis of PCR product.

OBJECTIVE:

 To demonstrate the amplification of discrete DNA fragments using PCR.

 To demonstrate the way to carry out PCR.
 To analyze the PCR product by electrophoresis.

INTRODUCTION:

DNA to be amplified is denatured by heating the sample in a thermal cycler. In the presence of
DNA polymerase and excess deoxynucleotide triphosphates, oligonucleotides that hybridize
specifically to the target sequence can prime new DNA synthesis. The first cycle is characterized
by a product of indeterminate length; however, the second cycle produces the discrete “short
product” which accumulates exponentially with each successive round of amplification. This can
lead to the many million-fold amplification of the discrete fragment over the course of 20 to 30
cycles.

APPARATUS AND MATERIALS:

Sterile H2O, 25 mM MgCl, 2 PCR microtubes, micropipettes & tips, 10X PCR buffer, 10 mM
dNTP mix, 10 µM oligonucleotides primers 1 and 2, template DNA, U Taq DNA polymerase,
sterile toothpicks, bacteria culture plates, automated PCR thermal cycler, agarose gel
electrophoresis apparatus.

METHODOLOGY:

1. Bacterial colonies on agar plate was transferred into a PCR tube using a toothpick.
2. Then, 25 µl of sterile distilled water was added and the mixture was boil for 10 minutes.
3. Mixture was then centrifuge for 5 minutes at 1000 rpm.
4. From the overall mixture, 5 µl will be used as template DNA.
5. A master mix was prepared as follow (for 1 reaction mixture):
 2.5 µl 10x amplification buffer
0.5 µl dNTP mix
1 µl oligonucleotide primer 1
1 µl oligonucleotide primer 2
Template DNA
Top up to 25 µl with sterile deionized H2O

6. Once the overall mixture is done, 0.5 µl of Taq DNA polymerase was added to the reaction
tube.
7. The reaction mixtures was then incubated in a pre-programmed automated PCR thermal
cycler.
8. Each sample is then run in an agarose gel to determine the success the PCR amplification.

DISCUSSIONS:

Polymerase Chain Reaction, PCR, is a procedure carry out to amplify DNA sequences. It will
make use the short sequences of DNA and primers and certain chromosome on the DNA will be
replicated. For this experiment, the source for PCR was taken from the bacterial colonies, E.coli,
which was prepared by the lab officer. After transferring the colonies into PCR tube whereby the
material undergoes boiling and centrifugation. The purpose incubation of the mixture that was
carried out for 10 minutes is to disintegrate the plasma membrane and also inactivate the DNAses
content which has a tendency of degrading the DNA content. After centrifugation, only the
supernatant is needed while the pellet was discarded as the pellet contains matrix bead that capable
of inhibiting the PCR procedure. Master mix was added as it is a mixture PCR reaction mix PCR
primer solution. This overall master mix is an important mixture for this PCR procedure as it
contain deoxyribonucleotide triphosphates mix, Taq DNA polymerase and Mg2+ ions.

Taq DNA polymerase is a type of enzyme that has high resistance towards high temperature and
it is used to synthesize new strands of DNA. The PCR primers which is short oligonucleotides
function is to allow complementary base pairing, one primer attaches to the top strand at one end
of your segment of interest, and the other primer attaches to the bottom strand at the other end.
Other than that, sterile deionized H20 was used as PCR is a highly sensitive procedure, thus, H20
that is free from DNA and RNA should be used.

Once placed in the PCR thermal cycler, forty cycles of amplification were done. Each cycle has
its own function and different temperature were applied. Three main steps in this PCR cycler is
denaturation, annealing and extension. Firstly, the temperature is set to 94°C for 1 minute with the
purpose of DNA denaturation into single strands. Then, 60°C for 1 minute to ensure primers
annealing on both sides of target sequence. Lastly, 72°C for 1 minute. This step is when Taq
polymerase bind and made a complimentary DNA strand to the single stranded DNA.

On the next session, gel electrophoresis was done to determine the success of the PCR. When the
PCR product viewed under the UV transiluminator, no bands was produced showing that the PCR
procedures that was carried out was not successful. During the viewing, it can be seen that there is
a clump of DNA stuck on the well. This is due to poor DNA purification skills thus the DNA
appear to be dirty. It may happen also since no washing with ethanol steps was done.

There are few factors that need can done to increase the yield of PCR product. One of it is to
increase the Mg2+ concentration. Other than that, maintain the dNTP concentration. As higher
concentration may leads to Mg2+ depletion occurs. Contamination the DNA or the oligonucleotide
primers itself may inhibit PCR. Thus, a proper purified or desalted primers should be used.
Concentration of primers used should also be moderate as high concentration of primer may causes
binding nonspecifically at undesirable sites and if possible only well-designed primers should be
used.

CONCLUSION:

The PCR product does not produce any bands when viewed under UV transilluminator indicating
that the procedure that was carried out was not successful. Therefore, if proper care was taken, it
can be ensure that proper yielding of PCR product can be produced.
REFRENCES:

1. Morales,N. (2017). PCR discussion. [online] Available at:

https://www.scribd.com/doc/46768464/PCR-Discussion [Accessed 28 March 2017].

2. Faculty.unlv.edu. (2017). PCR lab. [online] Available at:

https://faculty.unlv.edu/wmojica/PCR_LAB2.htm [Accessed 28 March 2017].

3. Chhetri,G. (2015). How can I improved the PCR output of low concentrated DNA? [online]
Available at:
https://www.researchgate.net/post/How_can_I_improve_the_PCR_output_of_a_low_con
centrated_DNA [Accessed 28 March 2017].

4. UK Essays. (2013). Polymerase Chain Reaction Lab Report Biology Essay. [online]
Emedicine.medscape.com. Available at:
https://www.ukessays.com/essays/biology/polymerase-chain-reaction-lab-report-biology-
essay.php [Accessed 28 March 2017].