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BIOLOGY | BIOTECHNOLOGY | DNA ANALYSIS METHODS

Gelelectrophoresis
A technique used to separate DNA fragments and other macromolecules by size and
charge.

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Key points:
Gel electrophoresis is a technique used to separate DNA fragments
according to their size.

DNA samples are loaded into wells (indentations) at one end of a gel, and
an electric current is applied to pull them through the gel.

DNA fragments are negatively charged, so they move towards the


positive electrode. Because all DNA fragments have the same amount of
charge per mass, small fragments move through the gel faster than large
ones.

When a gel is stained with a DNA-binding dye, the DNA fragments can be
seen as bands, each representing a group of same-sized DNA
fragments.

https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/a/gel-electrophoresis[22-03-2017 22:40:59]
Gel electrophoresis (article) | Khan Academy

Introduction
Suppose you have just done a PCR reaction, making many copies of a target
DNA region. Or perhaps youve done some DNA cloning, trying to "paste" a
gene into a circular DNA plasmid.

Now, you want to check and see whether your PCR worked, or whether your
plasmid has the right gene in it. What technique can you use to visualize
(directly observe) the fragments of DNA?

Gel electrophoresis
Gel electrophoresis is a technique used to separate DNA fragments (or
other macromolecules, such as RNA and proteins) based on their size and
charge. Electrophoresis involves running a current through a gel containing
the molecules of interest. Based on their size and charge, the molecules will
travel through the gel in different directions or at different speeds, allowing
them to be separated from one another.
[Where does the name "electrophoresis" come from?]

All DNA molecules have the same amount of charge per mass. Because of
this, gel electrophoresis of DNA fragments separates them based on size
only. Using electrophoresis, we can see how many different DNA fragments
are present in a sample and how large they are relative to one another. We
can also determine the absolute size of a piece of DNA by examining it next
to a standard "yardstick" made up of DNA fragments of known sizes. [More info]

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Gel electrophoresis (article) | Khan Academy

What is a gel?
As the name suggests, gel electrophoresis involves a gel: a slab of Jello-like
material. Gels for DNA separation are often made out of a polysaccharide
called agarose, which comes as dry, powdered flakes. When the agarose is
heated in a buffer (water with some salts in it) and allowed to cool, it will form
a solid, slightly squishy gel. At the molecular level, the gel is a matrix of
agarose molecules that are held together by hydrogen bonds and form tiny
pores.

At one end, the gel has pocket-like indentations called wells, which are
where the DNA samples will be placed:

Before the DNA samples are added, the gel must be placed in a gel box.
One end of the box is hooked to a positive electrode, while the other end is
hooked to a negative electrode. The main body of the box, where the gel is
placed, is filled with a salt-containing buffer solution that can conduct current.
Although you may not be able to see in the image above (thanks to my
amazing artistic skills), the buffer fills the gel box to a level where it just barely
covers the gel.

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Gel electrophoresis (article) | Khan Academy

The end of the gel with the wells is positioned towards the negative electrode.
The end without wells (towards which the DNA fragments will migrate) is
positioned towards the positive electrode.

How do DNA fragments move through the gel?


Once the gel is in the box, each of the DNA samples we want to examine (for
instance, each PCR reaction or each restriction-digested plasmid) is carefully
transferred into one of the wells. One well is reserved for a DNA ladder, a
standard reference that contains DNA fragments of known lengths.
Commercial DNA ladders come in different size ranges, so we would want to
pick one with good "coverage" of the size range of our expected fragments.

Next, the power to the gel box is turned on, and current begins to flow
through the gel. The DNA molecules have a negative charge because of the
phosphate groups in their sugar-phosphate backbone, so they start moving
through the matrix of the gel towards the positive pole. When the power is
turned on and current is passing through the gel, the gel is said to be
running. [What voltage is used to run a gel?]

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Gel electrophoresis (article) | Khan Academy

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Gel electrophoresis (article) | Khan Academy

Based on similar diagram in Reece et al.2

As the gel runs, shorter pieces of DNA will travel through the pores of the gel
matrix faster than longer ones. After the gel has run for awhile, the shortest
pieces of DNA will be close to the positive end of the gel, while the longest
pieces of DNA will remain near the wells. Very short pieces of DNA may have
run right off the end of the gel if we left it on for too long (something I've most
definitely been guilty of!).

Visualizing the DNA fragments


Once the fragments have been separated, we can examine the gel and see
what sizes of bands are found on it. When a gel is stained with a DNA-
binding dye and placed under UV light, the DNA fragments will glow, allowing
us to see the DNA present at different locations along the length of the gel.

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Gel electrophoresis (article) | Khan Academy

The bp next to each number in the ladder indicates how many base pairs
long the DNA fragment is.

A well-defined line of DNA on a gel is called a band. Each band contains a


large number of DNA fragments of the same size that have all traveled as a
group to the same position. A single DNA fragment (or even a small group of
DNA fragments) would not be visible by itself on a gel.

By comparing the bands in a sample to the DNA ladder, we can determine


their approximate sizes. For instance, the bright band on the gel above is
roughly 700 base pairs (bp) in size.

Check your understanding

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Gel electrophoresis (article) | Khan Academy

Four lanes are numbered on the gel above. (A lane is a corridor through
which DNA passes as it leaves a well.)

Which lane matches each description below?

1 2 3 4

This lane contains the longest DNA fragment.

This lane contains the shortest DNA fragment.

This lane contains a 1500 base pair (bp) DNA


fragment.

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Gel electrophoresis (article) | Khan Academy

[Hide hint]

In a DNA gel, the longest DNA fragments migrate most slowly through the
gel and stay closest to the wells (where the are initially loaded into the gel).
The shortest DNA fragments migrate most quickly through the gel and get
the closest to its far end (away from the wells).

Thus, the band representing the longest fragment will be closest to the top
of the gel. This band is found in lane 1.

Similarly, the band representing the shortest fragment will be closest to the
bottom of the gel. This band is found in lane 2.

We can use the DNA ladder (in the leftmost lane) to figure out which lane
contains a 1500 bp band. If we trace the 1500 bp band of the ladder
rightwards across the other lanes, we find a band of matching size in lane 3.

1 2 3 4

This lane contains the longest DNA fragment.

This lane contains the shortest DNA fragment.

This lane contains a 1500 base pair (bp) DNA


fragment.

Check

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Gel electrophoresis (article) | Khan Academy

[Attribution and references]

DNA analysis methods

Polymerase chain reaction (PCR)

Gel electrophoresis

Polymerase chain reaction (PCR)

Gel electrophoresis

DNA sequencing

Next tutorial
Stem cells

Questions
Tips & Thanks Top
Recent

Is it possible to make gel electrophoresis determination machine in home ?


2 votes



1 comment
Flag 10 months ago
by

Hassan Mohammad Alamin

I personally don't know, but if you Google search on "how to make your own gel box,"
some hits come up - maybe one of those would help you? Good luck! :)
3 votes



5 comments
Flag 10 months ago
by

emilyabrash

Show all 2 answers


Answer this question

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Gel electrophoresis (article) | Khan Academy

Are there more recently devloped methods to measure DNA length?


1 vote



1 comment
Flag 3 months ago
by

Forrest T

I would also add that researchers almost always use gel electrophoresis to at least
check that the PCR was successful as sending a failed PCR product to another lab for
sequencing etc. would be a complete waste of money and it's not cheap yet!
1 vote



Comment
Flag 2 months ago
by

Skylar Peven

How can we determine the longest or shortest DNA molecule of plasmid or a bacteria ? We
know that they are " super-coiled " and can move easily through the gel . this question is
hitting hard in my mind .... :)
1 vote



Comment
Flag 9 months ago
by

Mahmood shahid

I am not an expert but nobody else has answered you so here goes: I am pretty sure
you want to a restriction enzyme to clip the plasmid first. And perhaps you want to do
some other trick to uncoil it.
1 vote



Comment
Flag 3 months ago
by

John Morgenthaler

Why is electrophoresis run on constant power?


1 vote



Comment
Flag 2 months ago
by

sandhya07yadav

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Gel electrophoresis (article) | Khan Academy

which steps involve in gel electrophoresis?


1 vote



Comment
Flag 18 days ago
by

Learner

Which poles are known as the cathode and anode? Sorry I get a bit confused with these
two :\
1 vote



Comment
Flag 15 days ago
by

ruthpoh99

The cathode is negatively charged and the anode is positively charged.


1 vote



Comment
Flag 15 days ago
by

briancsherman

seems like a stupid question..but if you know the length of the samples using DNA ladder
as a scale , then how did the scientist knew the length of the first DNA ever measured?
sorry for my english btw.
1 vote



1 comment
Flag 5 months ago
by

Abda

When is each type of electrophoresis applicable? You mention that gel electrophoresis can
be used for RNA and proteins as well, however I am only finding examples using DNA. I
was always under the impression that gel electrophoresis is only used for DNA, and SDS-
PAGE is used for macromolecules like protein and RNA.
1 vote



Comment
Flag 21 days ago
by

J_G_

https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/a/gel-electrophoresis[22-03-2017 22:40:59]
Gel electrophoresis (article) | Khan Academy

Does the smear of bands of the PCR-reaction in the first picture in "Visualizing the DNA
fragments" have any meaning? How should it be interpreted?
1 vote



Comment
Flag 5 months ago
by

Joost Bus

Depending on how the PCR primers are selected they can sometimes
weakly/infrequently attach to other DNA sequences that are similar to the target
sequence. Then you will get some amplification of sequences that you did not mean to
target. This could be what is being seen.
1 vote



Comment
Flag 2 months ago
by

Skylar Peven

Show all 2 answers


Answer this question

Polymerase chain reaction (PCR) DNA sequencing

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Gel electrophoresis (article) | Khan Academy

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