Agarose Gel Electrophoresis Exam Questions and Answers Latest Update Graded A+
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Agarose Gel Electrophoresis
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Agarose Gel Electrophoresis
Agarose Gel Electrophoresis Exam Questions and Answers Latest Update Graded A+
what is Agarose gel electrophoresis? - Answers 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 electro...
Agarose Gel Electrophoresis Exam Questions and Answers Latest Update Graded A+
what is Agarose gel electrophoresis? - Answers 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? - Answers This is the standard method for identifying and
analyzing nucleic acids (DNA, RNA).
what is Agarose gel electrophoresis also known as? - Answers 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? - Answers 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? - Answers The loading dyes also contain nuclease inhibitors (EDTA)
what do nuclease inhibitors (EDTA) do? - Answers 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? - Answers 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? - Answers The DNA molecules are conducted through
the regularly sized pores of the gel.
The migration rates of nucleic acid molecules is determined by what? - Answers The migration rates of
nucleic acid molecules is determined by their size and conformation
Most gels are cast in the range of what? - Answers of 0.5 to 3.0%.
Higher gel percentages produce smaller or bigger pore sizes? - Answers 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? - Answers Lower percentage gels will have
larger pores and allow larger nucleic acids to migrate with relatively minimal frictional resistance.
what is agarose? - Answers 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? - Answers Loading/running dye mixture is added to
DNA or RNA samples in aqueous buffer.
how much of the dye will be added? - Answers 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? - Answers 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? - Answers
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? - Answers 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)? - Answers estimating the sizes of
DNA fragments typically involves running the unknown DNA against a set of standards of known sizes. A
good estimate of the sizes of the fragments is taken from a standard curve of the linear standards versus
distance migrated from the origin.
how are the DNA all running at the same position in the gel? - Answers The hundreds or thousands of
double-stranded DNA molecules in each band are all of the same size and configuration, which is why
they are running at the same position in the gel.
what was the original stain used until several years ago (and still used in some places)? - Answers
Ethidium bromide
what gel suspended the Ethidium bromide? - Answers Gel-Red (right), which has the same degree of
binding and staining sensitivity as EtBr, but is far less toxic and is now widely used instead of EtBr.
Both dyes are what shape and able to do what? - Answers Both dyes are planar (flat) and are able to
intercalate between the stacked base pairs of double-stranded DNA or RNA, or against single-stranded
RNA. The positive charge on the nitrogen (N+) allows the dye to bind to the nucleic acid's opposite
charged negative phosphates.
what third type of DNA staining dye will bind to DNA more or less in the same way as Gel-Red or
ethidium bromide (small positive charge; intercalating aromatic rings)? - Answers Sybr Green
DNA stained with Sybr Green can be visualised by UV excitation in the same way as the other dyes, but
there is a difference, what is it? - Answers the excitation spectrum of Sybr Green is at a higher
wavelength. The one property of Sybr Green that differs from the other dyes is that it does not bind as
well to single stranded DNA or RNA (fluorescence is 11x weaker).
how is the stained gel viewed? - Answers The stained gel is viewed on an ultraviolet (UV) light box with
the UV lamp set to irradiate the nucleic acids at a wavelength of 302 nm, whereupon the nucleic acids
absorb the UV light, fluoresce, and re-emit it at 540 nm in the red-orange visible region of the light
spectrum.
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