What is Gel Electrophoresis | Don't Memorise
Summary
TLDRThis script delves into the fundamental technique of gel electrophoresis, a method used to separate DNA molecules by size. It explains the process, starting from the negatively charged DNA's movement in an electrical field through an agarose gel, to the use of ethidium bromide for visualization under UV light. The script also covers the use of DNA ladders for size determination and the extraction of specific DNA fragments for further analysis, highlighting gel electrophoresis as a crucial tool in recombinant DNA technology.
Takeaways
- đ Gel electrophoresis is a technique used to separate DNA molecules based on their size.
- đ The term 'gel electrophoresis' comes from the use of an electrical field in a gel medium to separate charged molecules.
- đ DNA molecules are negatively charged due to the phosphate groups in their backbone, which facilitates their movement in the gel.
- đ Agarose gel, derived from seaweed, is commonly used as the medium for DNA separation.
- đŹ The separation principle is analogous to a sieve, where smaller particles pass through more easily than larger ones.
- 𧏠DNA samples with different sized fragments are loaded into wells in the gel for the electrophoresis process.
- đ An electric field is applied, causing the negatively charged DNA fragments to move towards the positive anode.
- đš A colored loading dye is used to visualize the movement of DNA through the gel, as DNA itself is colorless.
- đ Ethidium bromide is used to stain the DNA, allowing the separated fragments to be seen under ultraviolet light as orange bands.
- đ The size of the DNA fragments can be determined by comparing the position of the bands to a DNA ladder.
- đŹ The process of cutting out and extracting the desired DNA fragment from the gel is known as 'excision' and is used for further analysis.
Q & A
What is gel electrophoresis?
-Gel electrophoresis is a technique used to separate different DNA molecules based on their sizes by applying an electrical field in a gel medium.
Why is the technique called 'gel electrophoresis'?
-The term 'gel electrophoresis' comes from the fact that the separation of DNA molecules occurs within a gel medium under the influence of an electrical field.
What charge do DNA molecules carry?
-DNA molecules are negatively charged due to the presence of phosphate groups in their backbone, which are negatively charged.
What type of gel is commonly used for DNA separation in gel electrophoresis?
-Agarose gel, which is obtained from seaweeds, is commonly used for DNA separation in gel electrophoresis.
How does the size of DNA fragments affect their movement in the gel?
-Smaller DNA fragments move faster through the gel's pores, while larger fragments find it more difficult to move and lag behind due to the size of the pores and their own size.
What is the purpose of using a comb to create wells in the gel?
-The comb is used to create wells in the gel to load the DNA samples, allowing for the separation of DNA fragments when the electric field is applied.
Why are the wells for loading DNA samples formed near the cathode?
-The wells are formed near the cathode because the negatively charged DNA fragments will move towards the positively charged anode when the electric field is applied.
What is the role of the colored loading dye in gel electrophoresis?
-The colored loading dye is used to track the movement of the DNA through the gel, as DNA is colorless. It travels slightly faster than the DNA segments, indicating their progress.
How is the separated DNA visualized after electrophoresis?
-The separated DNA is visualized by treating the agarose gel with ethidium bromide, which binds to the DNA and fluoresces under ultraviolet light, revealing bright orange bands representing the DNA fragments.
How can the size of the DNA fragments be determined after separation?
-The size of the DNA fragments can be determined by comparing the positions of the bands on the gel with a standard DNA ladder, which provides a reference for fragment sizes.
What is the process called when the desired DNA fragment is cut out from the gel and extracted?
-The process of cutting out and extracting the desired DNA fragment from the gel is called 'excision', and the extracted DNA can be used for further downstream processing.
Outlines
đŹ DNA Fragment Separation through Gel Electrophoresis
This paragraph introduces the concept of gel electrophoresis, a technique used to separate DNA molecules based on size. It explains that DNA, being negatively charged due to phosphate groups, moves through an agarose gel under the influence of an electric field. The process is likened to a sieve, where smaller particles pass through more easily than larger ones. The paragraph also details the setup for gel electrophoresis, including the use of a casting tray, comb for wells, electric supply, and a DNA sample. It emphasizes the importance of the gel's mesh-like structure at a microscopic level, which allows for the separation of DNA fragments according to their size, with smaller fragments moving faster through the gel's pores.
đ Visualizing DNA Fragments Post-Electrophoresis
The second paragraph delves into the practical steps of conducting gel electrophoresis. It describes the preparation of the apparatus with a buffer for better electrical conductivity and the loading of the DNA mixture into the gel wells. The use of a colored loading dye is highlighted for tracking the movement of DNA through the gel. The paragraph explains that once power is applied, the DNA moves towards the positively charged anode, with the loading dye indicating the progress of the DNA's journey. Upon completion, the DNA fragments are made visible by treating the gel with ethidium bromide, which binds to DNA and fluoresces under ultraviolet light, revealing distinct bands corresponding to different DNA fragment sizes. The bands are then compared to a DNA ladder for size determination, and the desired fragments can be extracted from the gel for further use. The paragraph concludes by emphasizing the significance of gel electrophoresis in recombinant DNA technology and encourages viewers to stay informed about such scientific processes.
Mindmap
Keywords
đĄGel Electrophoresis
đĄDNA Fragments
đĄCharged Molecules
đĄAgarose Gel
đĄRestriction Enzymes
đĄSieve Analogy
đĄElectric Field
đĄLoading Dye
đĄBuffer
đĄEthidium Bromide
đĄDNA Ladders
đĄElution
Highlights
Introduction to gel electrophoresis as a technique for separating DNA molecules based on size.
Explanation of why the process is called gel electrophoresis, involving the movement of charged molecules in an electrical field within a gel medium.
DNA's negative charge due to phosphate groups in its backbone, influencing its movement in the gel.
Use of agarose gel, derived from seaweed, as the medium for DNA separation.
The principle of DNA fragment separation analogous to a sieve, with smaller particles moving faster through the gel's pores.
Description of the gel's microscopic mesh-like structure and its uniform pore size affecting DNA fragment mobility.
Setup requirements for gel electrophoresis, including a casting tray, gel, comb, electric supply, and DNA sample.
Importance of forming wells in the gel for loading DNA samples near the negative terminal or cathode.
Use of a colored loading dye to track the movement of DNA through the gel, which travels faster than the DNA fragments.
The role of a buffer in providing better conductivity for the electric field during the process.
Process of loading the DNA mixture into the wells and turning on the power supply to initiate DNA movement through the gel.
Observation of DNA movement cessation indicated by the loading dye reaching the anode.
Technique of treating the agarose gel with ethidium bromide to visualize DNA under ultraviolet light.
Identification of DNA bands as bright orange colors, representing separated DNA fragments of varying sizes.
Comparison of DNA fragment bands with a DNA ladder to determine the size of the fragments.
Process of excising and extracting the desired DNA fragment from the gel for further downstream processing.
Emphasis on gel electrophoresis as a crucial technique in recombinant DNA technology.
Invitation to stay tuned to the channel for more interesting scientific processes and to subscribe for updates.
Transcripts
[Music]
this kind of an image is not new to us
especially for people who love forensic
science but what exactly is this it's an
image of the separated DNA fragments on
a piece of gel and the process of
separating them is called gel
electrophoresis doesn't the sound very
difficult separating DNA fragments on a
piece of gel but actually the process of
gel electrophoresis is not very
difficult in fact it's quite easy let's
have a look at how the process works and
also the principle behind it let's begin
with the definition first gel
electrophoresis is a technique to
separate the different DNA molecules
based on their sizes can you guess why
is it called gel electrophoresis okay
let me help you with this this
separation technique is based on the
movement of charged molecules when
exposed to an electrical field and this
movement occurs in a gel medium hence
it's known as gel electrophoresis now
let's understand the process step by
step but before that since the term
charge is used by us here can you guess
the charge present on a DNA molecule
let me tell you that a DNA is negatively
charged and this is due to the presence
of phosphate groups these phosphate
being negatively charged and present in
the backbone of the DNA double helix
impart a negative charge to the complete
molecule and what is the gel used
interestingly the gel which is generally
used for the DNA is agarose gel and it's
obtained from the seaweeds now getting
back to a process we said that the DNA
molecules get separated on the basis of
their sizes so if we have a sample of
DNA which is containing fragments formed
with the activity of restriction enzymes
then separating the fragments based on
the difference in their sizes would be
possible with gel electrophoresis
let's have a look at this in detail do
you understand the principle and working
let's take the analogy of a sieve we
note that if a mixture of different
sized particles is poured onto a sieve
then only the particles smaller in size
than the pores will escape while those
bigger in size will be retained back
here the size of the particles is the
only parameter used for the separation
same is the case with DNA fragments when
our DNA sample containing various sized
fragments is loaded onto a gel then the
charge applied across the gel helps in
the mobility of these fragments however
not all fragments move at the same pace
those which are smaller move ahead and
those which are larger in size find it
difficult to move to understand this
better let's zoom into the gel that is
prepared for the experiment here at the
microscopic level we find that the gel
appears to be a mesh-like structure and
the pore size is nearly constant
throughout so now imagine what will
happen if different sized DNA fragments
are made to pass through these pores
it's obvious that the small sized
fragments will escape the pores faster
while the larger fragments will find it
difficult to come out they will take a
lot of time to cross the smaller pores
and dust will lag behind this is how we
can understand the principle of
separation of the DNA fragments based on
their sizes now let's see how the
procedure works the first requirement is
the complete set up this will include
the casting tray gel a comb for making
wells in the gel electric supply and
most importantly DNA sample with
different sized DNA fragments here's an
illustration of the casting tray
containing the gel in which we will load
the DNA mixture we usually opt for a
comb to form well like structures into
the gel and why do we do that because
it's in these worlds in the agarose gel
that we load the DNA mixture now tell me
where should the wells be formed at the
ends or at the centre think about it
we know that the DNA fragments being
negatively charged will travel from a
point of negative charge to the point of
positive charge
does we need to form the wells to load
the DNA samples near the negative
terminal or the cathode to be precise so
that the DNA will move towards the
positively charged anode this movement
of the DNA molecule will be promoted by
the electric field but we cannot track
the movement of the DNA in the gel as
it's colorless for this we use a colored
loading dye to track the movement of the
DNA the mixture of the DNA samples with
the colored loading dye is now all set
to get into the gel but before loading
the DNA mixture we will fill the
apparatus with a buffer as shown
the buffer is used to provide better
conductivity of electricity next we will
load the DNA mixture into the wells and
we're ready to turn on the power supply
once the power supply is turned on the
DNA mixture will move through the gel
which can be tracked with the help of
the loading dye the loading dye is
selected in such a way that it travels a
bit faster than the DNA segments present
in the mixture and why is that so that's
because we want the loading dye to reach
the terminal end faster than the DNA
once the dye reaches the anode we get an
indication that the DNA must have
reached somewhere near and does the
power supply has to be turned off
moving ahead with the next step now we
know that the DNA molecules have got
separated but can we really see them no
that's not possible so how will that be
possible now how do we observe the
separated segments to observe the DNA
molecules we treat the agarose gel with
helium bromide solution the major reason
for using ethidium bromide is that it
easily binds to the DNA molecules and
when the agarose gel containing the DNA
is observed under ultraviolet light
bright orange colored bands are clearly
seen these are nothing but the bands of
the DNA the separation has been
successful based on the size of the DNA
fragments the larger molecule are the
ones found here which means these are
the ones that moved slowly on the other
hand the smaller molecules which moved
faster are the ones
spotted here now how do we know the size
of any DNA fragment the bands obtained
are compared with a standard chart known
as the DNA ladders on comparing the
positions of the bands with the ones
from the chart we can easily make out
the length of the fragments obtained
once we know the length of the fragments
we can easily identify the desired DNA
now the desired DNA fragment can be
first manually cut out from the agarose
gel and then extracted this process is
called illusion the extraction of the
DNA is done in such a way that it can be
used for further downstream processing
this was the simple explanation of how
any gel electrophoresis technique works
it's considered as one of the very
important techniques used in recombinant
DNA technology to learn more about such
interesting processes stay tuned to our
Channel and do not forget to subscribe
happy learning
you
5.0 / 5 (0 votes)