Enhancing the Visualization of PCR Products by Troubleshooting of Gel Electrophoresis
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PCR Polymerase Chain Reaction, DNA fragment, gel electrophoresis, agarose gel, Taq polymerase, molecular biology, DNA extraction, DNA purity, PCR product, DNA ladder, blood sample, principle of PCR, DNA analysis, cell homeostasis, biological process, troubleshooting
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Abstract
Molecular biology is the study of biomolecules' mechanisms, assembly, and regulation of biological processes such as cell homeostasis. Many diseases can be diagnosed using molecular techniques such as PCR, microarray, western blotting, flow cytometry, and gel electrophoresis. PCR is a fascinating, versatile technique that has already established itself in biological and medical research and is the starting point for gaining information about the DNA molecule. In this report, we applied the steps of DNA analysis, which are extraction, master mix preparation, PCR, and gel electrophoresis. Furthermore, we have made an attempt to highlight the problems that occur during agarose gel electrophoresis, along with potential causes, and perform the troubleshooting.
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[...] Transfer the column to a new one after discarding the flow-through and tubular collection. add 500 µl of wash buffer I. centrifuge for 1 minute at 8000 rpm. Discard the collection tube and transfer the filter to a new one. Add 500 µl wash buffer II. Centrifuge for 3 minutes at 13000 rpm. Discard the collection tube and transfer the filter to a new 1.5-ml microfuge tube. Add 200 µl of elution buffer (wait 1-5 min). Centrifuge for 1 minute at 8000 rpm (Figure 1). [...]
[...] In (figure the results show gel electrophoresis for the DNA of 18 samples. All samples showed the same molecular weight, which is 180bp, with a 100bp ladder or marker which is a series of DNA fragments of known sizes that are used as a reference to calculate DNA fragments' sizes, which are unknown.Lanes and 4 gave good results, but lanes 5 - 19 showed smeared results. In ( Figure , The result showed that lane 7 is negative. The rest of the lanes gave good results, but they differ in molecular weight. [...]
[...] While 2 samples show poor resolution of DNA fragment results due to improper agarose gel concentration, which represents samples gave a good outcome, representing 66.03%. The problems occur during agarose gel electrophoresis in this study were 33.93%. It is a high percentage that we performing troubleshooting to minimize and limit the problems. V. Conclusion In this study, an attempt was made to highlight the problems that occur during agarose gel electrophoresis along with potential causes and perform troubleshooting. Most of the results were good visualization, some results show problems occur during gel electrophoresis. [...]
[...] Many diseases can be diagnosed using molecular techniques such as PCR, microarray, western blotting, flow cytometry, and gel electrophoresis. PCR is a fascinating, versatile technique that has already established itself in biological and medical research and is the starting point for gaining information about the DNA molecule. In this report, we applied the steps of DNA analysis, which are extraction, master mix preparation, PCR, and gel electrophoresis. Furthermore, we have made an attempt to highlight the problems that occur during agarose gel electrophoresis, along with potential causes, and perform the troubleshooting. [...]
[...] Int J Biomed Res. 2011;2(1). doi:10.7439/ijbr.v2i1.83 9. Valones MAA, Guimarães RL, Brandão LAC, de Souza PRE, de Albuquerque Tavares Carvalho Crovela S. Principles and applications of polymerase chain reaction in medical diagnostic fields: a review. Braz J Microbiol. 2009;40(1):1-11. [...]
