Enhancement the Visualization of PCR
Products by Troubleshooting of Gel
Electrophoresis
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.
Introduction:
Molecular biology is the field of life science concerned with studying basic molecular components of single cells.
Including the biomolecules structure, characteristics, and function2. Furthermore, molecular biology in its most
basic form is the information that DNA stores before being transcribed into RNA and then translated into
proteins3.Molecular biology specifically deals with DNA, RNA, and proteins that are built using the genetic
instructions encoded in those molecules1. There are many different techniques used in molecular biology, such
as polymerase chain reaction (PCR), microarray analysis, and western blotting. PCR has progressed from a highly
specialized research tool to a routine technique used in many medical laboratories4. Moreover, PCR reaction
generates a DNA molecule's designated area, which is amplified specifically. The properties of PCR make it
extremely versatile4. There are many clinical applications for PCR, such as the detection of mutations, the usage
of primers customized to a disease-causing virus's DNA, and the use of PCR with forensic DNA materials8,9.
Furthermore, there are different types of PCR: quantitative and qualitative. Real-time PCR, known as qPCR, is
used to amplify, quantify, and examine the products by using fluorescent dyes while the reaction is taking place7.
Qualitative PCR is conventional PCR, which is carried out in a tube, and once the reaction is finished, the
reaction's byproducts (the amplified DNA fragments) are examined and seen using gel electrophoresis5. Gel
electrophoresis uses either agarose or polyacrylamide gel. Separation of the PCR product via gel electrophoresis
according to the size of the molecule and charge4. The amplified DNA fragment can be observed as a discrete
band after staining with ethidium bromide (EthBr). Bands showing the position of the different size classes of
DNA fragments are clearly visible under ultraviolet irradiation after EthBr staining9.
, Material and Method:
Extraction of DNA from blood sample:
Based on the cross-sectional study, 53 blood samples were collected in an EDTA tube. Add 200 µl from each
blood sample into 1.5 ml microfuge tube. 200 µl of lysis buffer added then sample lysed by adding 20 µl of
protease/ proteinase K. After that the lysate undergo vortex and centrifugation (short spin) then incubate in water
bath at 56ºC for 10 min. then add 200 µl of absolute ethanol. Vortex and centrifuge (short spin) then transfer the
full amount to 2 ml collection tube was filled with a DNeasy Mini spin column. Centrifuge at 8000 rpm for 1
minute. 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). Measure the concentration and purity of extracted DNA using the Nanodrop machine
(Figure 2).
Products by Troubleshooting of Gel
Electrophoresis
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.
Introduction:
Molecular biology is the field of life science concerned with studying basic molecular components of single cells.
Including the biomolecules structure, characteristics, and function2. Furthermore, molecular biology in its most
basic form is the information that DNA stores before being transcribed into RNA and then translated into
proteins3.Molecular biology specifically deals with DNA, RNA, and proteins that are built using the genetic
instructions encoded in those molecules1. There are many different techniques used in molecular biology, such
as polymerase chain reaction (PCR), microarray analysis, and western blotting. PCR has progressed from a highly
specialized research tool to a routine technique used in many medical laboratories4. Moreover, PCR reaction
generates a DNA molecule's designated area, which is amplified specifically. The properties of PCR make it
extremely versatile4. There are many clinical applications for PCR, such as the detection of mutations, the usage
of primers customized to a disease-causing virus's DNA, and the use of PCR with forensic DNA materials8,9.
Furthermore, there are different types of PCR: quantitative and qualitative. Real-time PCR, known as qPCR, is
used to amplify, quantify, and examine the products by using fluorescent dyes while the reaction is taking place7.
Qualitative PCR is conventional PCR, which is carried out in a tube, and once the reaction is finished, the
reaction's byproducts (the amplified DNA fragments) are examined and seen using gel electrophoresis5. Gel
electrophoresis uses either agarose or polyacrylamide gel. Separation of the PCR product via gel electrophoresis
according to the size of the molecule and charge4. The amplified DNA fragment can be observed as a discrete
band after staining with ethidium bromide (EthBr). Bands showing the position of the different size classes of
DNA fragments are clearly visible under ultraviolet irradiation after EthBr staining9.
, Material and Method:
Extraction of DNA from blood sample:
Based on the cross-sectional study, 53 blood samples were collected in an EDTA tube. Add 200 µl from each
blood sample into 1.5 ml microfuge tube. 200 µl of lysis buffer added then sample lysed by adding 20 µl of
protease/ proteinase K. After that the lysate undergo vortex and centrifugation (short spin) then incubate in water
bath at 56ºC for 10 min. then add 200 µl of absolute ethanol. Vortex and centrifuge (short spin) then transfer the
full amount to 2 ml collection tube was filled with a DNeasy Mini spin column. Centrifuge at 8000 rpm for 1
minute. 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). Measure the concentration and purity of extracted DNA using the Nanodrop machine
(Figure 2).
