Agarose Gel Electrophoresis: Questions/Answers
(100%)
What is Agarose gel electrophoresis? Right Ans - A most fundamental
method in molecular biology, used to visualize and analyse nucleic acids in
many guises from many different methodologies.
What does Agarose gel electrophoresis do? Right Ans - This is the standard
method for identifying and analyzing nucleic acids (DNA, RNA).
What is Agarose gel electrophoresis also known as? Right Ans - It is also
known as submarine gel electrophoresis as the gel is submerged beneath the
running buffer.
The nucleic acid samples are loaded into what? Right Ans - The nucleic acid
samples are loaded in special dye mix that contains marker dyes to indicate
the extent of the run - these dyes run ahead of bromophenol blue, BPB or with
xylene cyanol nucleic acids of various sizes.
The loading dyes also contain what? Right Ans - The loading dyes also
contain nuclease inhibitors (EDTA)
What do nuclease inhibitors (EDTA) do? Right Ans - they prevent digestion
of DNA during the run, and also contain a percentage of sucrose or a high MW
polymer to give the sample dye mix viscosity so that it will settle and remain
in the well until the run is started.
Gels run until what? Right Ans - Gels are run until the BPB dye reaches at
least halfway or near the end of the gel.
The DNA molecules are conducted through what? Right Ans - The DNA
molecules are conducted through the regularly sized pores of the gel.
The migration rates of nucleic acid molecules is determined by what? Right
Ans - The migration rates of nucleic acid molecules is determined by their size
and conformation
, Most gels are cast in the range of what? Right Ans - of 0.5 to 3.0%.
Higher gel percentages produce smaller or bigger pore sizes? Right Ans -
Higher gel percentages produce smaller pore sizes and will only allow ready
migration of small fragments.
Lower percentage gels will have larger or smaller pores? Right Ans - Lower
percentage gels will have larger pores and allow larger nucleic acids to
migrate with relatively minimal frictional resistance.
What is agarose? Right Ans - Agarose is a purified component of agar that
melts at 100 degrees Celsius and sets (~45oC) in the same way as an agar.
Loading/running dye mixture is added into what? Right Ans -
Loading/running dye mixture is added to DNA or RNA samples in aqueous
buffer.
How much of the dye will be added? Right Ans - Typically, about 5 µl dye to
15 µl sample. The loading dye mixture is supplied or made at ~5x strength so
only a few µls are required per sample
What does the Agarose gel electrophoresis contain? Right Ans - It contains
buffer, EDTA, bromophenol blue dye (and often a second dye xylene cyanol,
which is green and runs slower than bromophenol blue), and something to
make the dye mix viscous, usually 30% sucrose or a high MW polysaccharide.
When the sample is added to the well via a pipette tip it drops into the well,
why is that? Right Ans - because it is more dense than the running buffer;
and it remains there until the current is applied.
The gel photo at the bottom of this slide (slide four number 3) depicts what?
Right Ans - The gel photo at the bottom of this slide depicts standards in the
left lane and decreasing amounts of the same sample in lanes 1 to 6. The point
to notice about these samples is the way the broadness of the bands bias the
actual size of the DNA sample. Although not a precise issue, the actual size of
the sample band is best taken from lane 6.
How does estimating the sizes of DNA fragments go about (slide 5)? Right
Ans - estimating the sizes of DNA fragments typically involves running the
(100%)
What is Agarose gel electrophoresis? Right Ans - A most fundamental
method in molecular biology, used to visualize and analyse nucleic acids in
many guises from many different methodologies.
What does Agarose gel electrophoresis do? Right Ans - This is the standard
method for identifying and analyzing nucleic acids (DNA, RNA).
What is Agarose gel electrophoresis also known as? Right Ans - It is also
known as submarine gel electrophoresis as the gel is submerged beneath the
running buffer.
The nucleic acid samples are loaded into what? Right Ans - The nucleic acid
samples are loaded in special dye mix that contains marker dyes to indicate
the extent of the run - these dyes run ahead of bromophenol blue, BPB or with
xylene cyanol nucleic acids of various sizes.
The loading dyes also contain what? Right Ans - The loading dyes also
contain nuclease inhibitors (EDTA)
What do nuclease inhibitors (EDTA) do? Right Ans - they prevent digestion
of DNA during the run, and also contain a percentage of sucrose or a high MW
polymer to give the sample dye mix viscosity so that it will settle and remain
in the well until the run is started.
Gels run until what? Right Ans - Gels are run until the BPB dye reaches at
least halfway or near the end of the gel.
The DNA molecules are conducted through what? Right Ans - The DNA
molecules are conducted through the regularly sized pores of the gel.
The migration rates of nucleic acid molecules is determined by what? Right
Ans - The migration rates of nucleic acid molecules is determined by their size
and conformation
, Most gels are cast in the range of what? Right Ans - of 0.5 to 3.0%.
Higher gel percentages produce smaller or bigger pore sizes? Right Ans -
Higher gel percentages produce smaller pore sizes and will only allow ready
migration of small fragments.
Lower percentage gels will have larger or smaller pores? Right Ans - Lower
percentage gels will have larger pores and allow larger nucleic acids to
migrate with relatively minimal frictional resistance.
What is agarose? Right Ans - Agarose is a purified component of agar that
melts at 100 degrees Celsius and sets (~45oC) in the same way as an agar.
Loading/running dye mixture is added into what? Right Ans -
Loading/running dye mixture is added to DNA or RNA samples in aqueous
buffer.
How much of the dye will be added? Right Ans - Typically, about 5 µl dye to
15 µl sample. The loading dye mixture is supplied or made at ~5x strength so
only a few µls are required per sample
What does the Agarose gel electrophoresis contain? Right Ans - It contains
buffer, EDTA, bromophenol blue dye (and often a second dye xylene cyanol,
which is green and runs slower than bromophenol blue), and something to
make the dye mix viscous, usually 30% sucrose or a high MW polysaccharide.
When the sample is added to the well via a pipette tip it drops into the well,
why is that? Right Ans - because it is more dense than the running buffer;
and it remains there until the current is applied.
The gel photo at the bottom of this slide (slide four number 3) depicts what?
Right Ans - The gel photo at the bottom of this slide depicts standards in the
left lane and decreasing amounts of the same sample in lanes 1 to 6. The point
to notice about these samples is the way the broadness of the bands bias the
actual size of the DNA sample. Although not a precise issue, the actual size of
the sample band is best taken from lane 6.
How does estimating the sizes of DNA fragments go about (slide 5)? Right
Ans - estimating the sizes of DNA fragments typically involves running the
