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Lab 88

The document outlines the procedures for performing conventional PCR amplification of cDNA samples and subsequent gel electrophoresis to verify the presence of the target gene. It details the principles of PCR, including denaturation, annealing, and extension, as well as the steps for preparing and running gel electrophoresis. Results indicate potential DNA degradation in some samples and the presence of the target gene in well number 2, with additional bands likely representing primer dimers.

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13 views4 pages

Lab 88

The document outlines the procedures for performing conventional PCR amplification of cDNA samples and subsequent gel electrophoresis to verify the presence of the target gene. It details the principles of PCR, including denaturation, annealing, and extension, as well as the steps for preparing and running gel electrophoresis. Results indicate potential DNA degradation in some samples and the presence of the target gene in well number 2, with additional bands likely representing primer dimers.

Uploaded by

A Th
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Lab 8

Aim:

1-performing conventional PCR amplification of the cDNA samples

2-Obtaining gel electrophoresis to make sure that the target gene is present and
been amplified.

Principle:

1-Conventional PCR

The Polymerase Chain Reaction (PCR) is a widely used molecular biology


technique developed by Kary Mullis in the 1980s. The method amplifies a specific
segment of DNA through repeated cycles of denaturation, annealing, and extension
using DNA polymerase. Here's a breakdown of the PCR principle:
Denaturation: The first step involves heating the DNA sample to around 94-98°C,
causing the double-stranded DNA to separate into two single strands. This process
breaks the hydrogen bonds between the complementary bases, resulting in the
formation of two single-stranded DNA molecules.
Annealing: The temperature is lowered to around 50-65°C. In this step, short DNA
primers, which are complementary to sequences flanking the target DNA region,
anneal (bind) to their complementary sequences on the single-stranded DNA
template.
Extension: The temperature is raised to around 72°C, the optimal temperature for
most DNA polymerases. DNA polymerase enzyme extends the primers by adding
nucleotides to the 3' end, synthesizing a new DNA strand complementary to the
template strand.
These three steps constitute one cycle of PCR. The process is then repeated for
multiple cycles, typically 20-40 times, exponentially amplifying the targeted DNA
sequence.
2-Gel electrophoresis:

1-Preparation of the Gel: A gel made of agarose is used for DNA electrophoresis.
(Agarose is a polysaccharide extracted from seaweed that forms a gel matrix with
small pores when solidified). The concentration of agarose can be adjusted to
optimize separation based on the size range of DNA fragments being analyzed.
2-Loading the Samples: The DNA samples are mixed with a loading buffer that
contains tracking dyes and a densifying agent to help the samples sink into the
wells of the gel. These samples are loaded into wells at one end of the gel using a
micropipette.
3-Application of Electric Field: Once the samples are loaded, an electric field is
applied across the gel by connecting the ends of the gel chamber to a power supply.
DNA is negatively charged due to its phosphate backbone, so it migrates towards
the positively charged electrode (anode).
4-Separation Based on Size: As the DNA molecules move through the gel matrix
under the influence of the electric field, smaller DNA fragments migrate more
quickly through the gel than larger ones because they encounter less resistance
from the gel matrix. This separation based on size allows DNA fragments of
different lengths to be resolved into distinct bands along the length of the gel.

References

1-Saiki, R. K., et al. (1988). Primer-directed enzymatic amplification of DNA with


a thermostable DNA polymerase. Science, 239(4839), 487-491.

2-Garibyan, Lilit, and Nidhi Avashia. "Research Techniques Made Simple:


Polymerase Chain Reaction (PCR)." Journal of Investigative Dermatology.

Principle:

1- PCR sample preparation:


1-0.5µl of forward GAPDH human primer (because our sample was from the
MDA cell line that wat treated with the datura extract + methotrexate).
2-0.5µl of reverse GAPDH human primer.
3-10 µl of the master mix.
4-8 µl nuclease free water.
5-1 µl of cDNA.

2-PCR thermal profile


1.Initial denaturation: 95°C, for 3min.
2.Denaturation: 95°C, for 30 sec.
3.Annealing: 55°C, for 1min.
4.Initial extension: 72°C, for 1min.
5.Final extension: 72°C, for 6min
6.Repeat step 2-5 for 40 cycles
7.Store for 24hr
Gel electrophoresis sample preparation:
1-Mix 100mL of 1xr TAE with 2g of agarose powder in a flask or other
microwaveable glassware.
2-Heat the mixture in the microwave until it boils, then pause and swirl it several
times. Do not let the contents boil over in the microwave. Once the fluid is clear
and the agarose is dissolved, remove it from the microwave and allow it to cool
slightly.
3-Once the solution has cooled slightly, pour it into the gel box.
4-Allow 20-30 minutes for the gel to set before removing the comb.
5-Cut a piece of parafilm and create parafilm "dots". We loaded the PCR output
directly in to the gel as it already contains a dye that was present in the master mix,
the volume loaded was between (15-20µl).
6-Place the gel tray into the gel rig and cover the gel with enough amount of 1X
TAE buffer.
7-Set the gel to run at 75v for 30-45min and begin the run.

Results

A B C D E F

Our target gene is expected to be approximately 1176 base pairs in length.


Notably, there appears to be evidence of DNA degradation in well numbers 1
and 2, which could have resulted from mishandling during preparation.
Furthermore, beyond the ladder region at the end of the gel, visible bands are
noticeable, likely attributed to the presence of primer dimers. Interestingly, in
well number 2, a distinct band is observable, potentially representing our
target gene.

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