Gel Electrophoresis (Article) - Khan Academy

4/18/2018 Gel electrophoresis (article) | Khan Academy
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BIOLOGY BIOTECHNOLOGY
SCIENCE · BIOLOGY · BIOTECHNOLOGY · DNA ANALYSIS
DNA analysis methods METHODS
Polymerase chain
Gel electrophoresis
reaction (PCR) A technique used to separate DNA fragments and other
macromolecules by size and charge.
Gel electrophoresis
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reaction (PCR)
Gel electrophoresis Key points:

Gel electrophoresis is a technique used to
DNA sequencing
separate DNA fragments according to their size.
Practice: DNA analysis

methods DNA samples are loaded into wells
(indentations) at one end of a gel, and an
Next tutorial electric current is applied to pull them through
Stem cells
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 1/14
Introduction
Suppose you have just done a PCR reaction, making
many copies of a target DNA region. Or perhaps
you’ve 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]
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
akes. 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 lled 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 lls the gel box to a level where it just barely
covers the gel.
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 ow 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?]
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 de nitely 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.
The bp next to each number in the ladder indicates how many base pairs
long the DNA fragment is.
A well-de ned “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
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.
[Hint]
Check
[Attribution and references]
Ask a question...
Questions Tips & Thanks Top Recent
Is it possible to make gel electrophoresis determination

machine in home ?
8 votes • 3 comments • Flag
2 years 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! :)
• 12 votes • 5 comments • Flag
2 years ago by emilyabrash
Show all 3 answers • Answer this question
Which poles are known as the cathode and anode? Sorry I get
a bit confused with these two :\
about a year ago by ruthpoh99
The cathode is negatively charged and the anode is

positively charged.
4 votes • Comment • Flag
about a year ago by briancsherman
Are there more recently devloped methods to measure DNA

length?
3 votes • 1 comment • Flag
about a year 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!
about a year ago by Skylar Peven
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 rst DNA ever measured?
sorry for my english btw.
about a year ago by Abda Shaffan
x-ray crystallography i believe was one of the rst

methods used to deduce DNA structure and length at
the turn of the 20th century; gel electrophoresis is
merely a convenience to compare known length to
unknown lengths of DNA, RNA and proteins.
10 months ago by conner.fundak
why do the bands appear to be of the same size while the

DNA fragments vary in their sizes?
3 votes • Comment • Flag about a year ago by Sualeha
The bands that you see are as a result of loading dye,

which helps scientists see the DNA they're loading into
the gel. The DNA fragments are typically illuminated
under UV light, and aren't visible in visible light.
about a year ago by City Face
what does it mean to have multiple bands for same sample

(for ex: sample#3 above). Also when two or more bands
appear for the same sample, which band do we use to
determine the size?
10 months ago by Kamini Joshi
Multiple bands mean DNA fragments with different size

and lengths. Realistically when doing gel electrophoresis
you'll see many more bands for the same sample. To
determine the bp size, you estimate using the reference
DNA.
1 vote • Comment • Flag
2 days ago by Michelle Ly
If the wells were placed in the middle of the gel as opposed to

the end, how would this allow us to infer information about
the charge of a macromolecule, as well as its size?
1 vote • Comment • Flag 3 months ago by sp0915
What force causes the DNA to move through the gel?

How would that change if the molecule had a positive vs.
a negative charge?
Does that help?

about a month ago by tyersome
How do we read the results? I know I did this lab in my

biology class at my school, but I've forgotten how to read it.
For example, which bands represents the trait/allele?
1 vote • Comment • Flag 10 months ago by Rippy
It depends a bit on the type of analysis and the size of

the allele you're looking at. In general, the larger allele
will be more toward where you loaded samples in the
gel. Agarose gels have uniformly sized pores in them, so
it takes larger stretches of sample longer to move
through it, and so it'll move slower through the gel than
something smaller.
1 vote • 1 comment • Flag
10 months ago by City Face
Why gel matrix pores have uniform size??

2 months ago by Shahzeen Shahbaz
Are there different types of DNA stains? If so, how do they

differ?
Do they all make the DNA glow or can they just make it a
color visible in UV light?
1 vote • Comment • Flag 6 months ago by Maggie
There are many, both uorescent and non uorescent.

Fluorescent is more sensitive. Some produce standard
visible light color (usually dark bluish), and some can be
used before electrophoresis, after electrophoresis or
both. Even Methylene Blue the commonest A level stain
can be used.
3 months ago by Alex Lain
Show more comments
Polymerase chain reaction (PCR) DNA sequencing
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Gel electrophoresis Key points:

Practice: DNA analysis

DNA fragments are negatively charged, so they

When a gel is stained with a DNA-binding dye,

Now, you want to check and see whether your PCR

All DNA molecules have the same amount of charge

DNA by examining it next to a standard "yardstick"

At one end, the gel has pocket-like indentations

Before the DNA samples are added, the gel must be

to a negative electrode. The main body of the box,

The end of the gel with the wells is positioned

How do DNA fragments move

Next, the power to the gel box is turned on, and

phosphate groups in their sugar-phosphate

Based on similar diagram in Reece et al.2

As the gel runs, shorter pieces of DNA will travel

Visualizing the DNA fragments

A well-de ned “line” of DNA on a gel is called a

By comparing the bands in a sample to the DNA

Check your understanding

Four lanes are numbered on the gel above. (A lane is

Which lane matches each description below?

This lane contains the

This lane contains the

This lane contains a 1500

[Attribution and references]

Questions Tips & Thanks Top Recent

Is it possible to make gel electrophoresis determination

I personally don't know, but if you Google search on

Show all 3 answers • Answer this question

The cathode is negatively charged and the anode is

Are there more recently devloped methods to measure DNA

I would also add that researchers almost always use gel

Show all 2 answers • Answer this question

seems like a stupid question..but if you know the length of the

x-ray crystallography i believe was one of the rst

Show all 2 answers • Answer this question

why do the bands appear to be of the same size while the

The bands that you see are as a result of loading dye,

what does it mean to have multiple bands for same sample

Multiple bands mean DNA fragments with different size

If the wells were placed in the middle of the gel as opposed to

1 vote • Comment • Flag 3 months ago by sp0915

What force causes the DNA to move through the gel?

Does that help?

How do we read the results? I know I did this lab in my

It depends a bit on the type of analysis and the size of

Why gel matrix pores have uniform size??

Are there different types of DNA stains? If so, how do they

There are many, both uorescent and non uorescent.

Show more comments

Polymerase chain reaction (PCR) DNA sequencing

Biology is brought to you with support from the

About Our team Our leadership

Impact Our content Our contributors

Press Test prep

Download our apps

© 2018 Khan Academy Terms of use Privacy notice

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