Theory and Introduction
Gel electrophoresis of
PCR products is a standard way of determining the quality of a reaction as well
as its yield. It should be noticed that PCR products range up to 9 kb in
length, and most of the amplifications remain consistent at 1kb where PAGE
analysis is considered a far better and more effective option than the others
(Berg, 2012). These days, gel electrophoresis is being widely used for
separating and analyzing a variety of macromolecules such as proteins, RNA and
DNA. With this technique, one can easily divide macromolecules into
micro-molecules for further studies (Williams & Goodeve). This method is
primarily used in clinical chemistry and helps separate proteins based on their
size or charge. In the fields of molecular biology and biochemistry, gel
electrophoresis is used to separate mixed populations of RNA and DNA fragments
and to estimate the actual size of each RNA or DNA fragment. The purpose of
this lab experiment was to visualize the given sample with UV light. No extra
staining was used for this purpose; in fact, the DNA stain was clearly visible.
Multiple bands from the PCR reaction indicated varying priming sites for the
primers of the target DNA.
Apparatus and Chemicals Used
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Materials Required for Advance Preparation
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Quantity
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UView 6x loading dye
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1 vial
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PCR molecular weight ruler
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1 vial
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Sterile water
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1 bottle
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PCR products for electrophoresis from
previous lesson
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4 per group
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2 ml microcentrifuge tubes
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24
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Materials Required but Not Supplied
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Quantity
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2–20 µl adjustable-volume micropipets
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8
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2–20 µl pipet tips, aerosol barrier
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8 racks
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50x TAE
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1 bottle
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Molecular biology grade agarose
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1 bottle
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Graduated cylinders, 3 L and 500 ml
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1
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Microwave or magnetic hot plate and stir bar
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1
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Bottle or Erlenmeyer flask, 1 L
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1
|
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Water bath at 60°C (optional)
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1
|
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Gel casting trays
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8
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Gel combs
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8
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Lab tape for gel casting (optional)
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1 roll
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Horizontal electrophoresis chamber
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8
|
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UV transilluminator or imaging system
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1
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Marking pens
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8
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Power supply
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2–4
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Procedure
1.
The PCR molecular weight ruler
was inserted in a centrifuge along with an aim to bring all the content to the
bottom.
2.
In the next step, we added 50
µl of UView 6x loading dye to the vial of PCR molecular weight ruler and mixed
everything nicely.
3.
Then we labeled the eight 2 ml
microcentrifuge tubes MWR and added 25 µl of PCR molecular weight ruler to each
of them.
4.
The eight 2 ml microcentrifuge
tubes were labeled Loading Dye, and we added 15 µl of UView 6x loading dye to
every tube.
5.
The next eight 2 ml
microcentrifuge tubes were labeled Sterile Water, and we then added 40 µl of
sterile water to every tube
|
|
Discussion
In this experiment, we
found that the genomic DNA had produced visible PCR products with the correct
size. The presence of human “transgene” sequence was observed during this
experiment. By looking at the graph, it becomes evident that there is a strong
relationship between the molecular weight of DNA and the distance it travels
(Charlene et al. 2012).
Conclusion
It has now been proved
that PCR can be used to study DNA and RNA fragments and that gel
electrophoresis is an effective method to analyze the quality and yield of PCR
products.
References
Berg, H. (2012). Restriction
Fragment Length Polymorphism Analysis of PCR-Amplified Fragments (PCR-RFLP) and
Gel Electrophoresis - Valuable Tool for Genotyping and Genetic
Fingerprinting. Gel Electrophoresis - Principles and Basics.
doi:10.5772/37724
Williams, I. J., &
Goodeve, A. C. (n.d.). Conformation-Sensitive Gel Electrophoresis. PCR
Mutation Detection Protocols, 137-150. doi:10.1385/1-59259-273-2:137
Charlene, R., Lori, M., John,
B., & Lari, M. (2012). Application of Multiplex PCR, Pulsed-Field Gel
Electrophoresis (PFGE), and BOX-PCR for Molecular Analysis of
Enterococci. Gel Electrophoresis - Principles and Basics.
doi:10.5772/37897
