Download Introduction to gel electrophoresis

Gel Electrophoresis
Gel Electrophoresis
• Gel electrophoresis
is used in order to
separate, identify,
and purify 0.5 to
25Kb DNA fragments.
• DNA has negatively
charged phosphates
along the DNA
backbone.
1
Gel Electrophoresis
• DNA fragments can be
separated by size when
applied to an electric field.
• DNA molecules migrate
toward the anode (+).
Gel Electrophoresis
• Agarose is a porous gelatinous
carbohydrate.
• The DNA samples are loaded
into an agarose gel mold.
• The agarose mold is placed into
a tank which contains a buffer
solution (TAE or TBE)
• The gel is run at a voltage and
for a time period that will
separate the DNA fragments
-
+
2
Gel electrphoresis
Gel Electrophoresis
• Large fragments of DNA
move slowly through the
agarose while small DNA
fragments move quickly.
3
Gel Electrophoresis
• Low agarose concentrations.
– 0.3 to 0.5%
– Separate large DNA fragments.
• 20 to 60kb
• Medium agarose concentrations.
– 0.5% to 1.0%
– Separate medium sized fragments.
• 0.5 to 30kb
• High agarose concentrations.
– 1 to 1.5%
– Separate small DNA fragments.
• 0.2 to 0.5kb
Gel Electrophoresis
• Molecular weight marker or
ladder - DNA fragments of
known sizes.
• Loaded into a separate lane.
• Sizes of unknown DNA
fragments can be estimated
using a molecular weight
marker.
4
Gel Electrophoresis
• DNA cannot be seen during gel
electrophoresis.
• Tracking Dye/Sample
Buffer/Loading Dye - used in
order to estimate how far the
DNA has run during a gel
electrophoresis.
• Negatively charged.
• Added to each DNA sample.
• Does not bind to the DNA!
Gel Electrophoresis
• Bromphenol Blue.
– Migrates with DNA
fragments around 0.5kb.
• Xylene Cyanol
– Xylene Cyanol migrates
with DNA fragments
around 5kb.
• Glycerol - Increases the
density of the sample for
easier loading.
Xylene
BMB
5
Gel Electrophoresis
• Between 5 and 200 ng of a
single DNA fragment should
be loaded into a well.
• Question  Are we
overloading our gels?
Not
overloaded
Overloaded
– We digested 10ul of DNA at
about .1ug/ul.
– How many ug did we digest?
– The entire amount of each
digested sample will be added
to each well.
– >200ng of DNA is considered
overloading.
Gel Electrophoresis
• Ethidium Bromide
(EtBr) - Used in order to
visualize the DNA.
• Intercalates (inserts)
between DNA’s base
pairs
• Fluoresces under UV
light
6
Gel Electrophoresis
• Longer DNA fragments will
contain more ethidium
bromide and appear darker
• Shorter DNA fragments will
contain less ethidium
bromide and appear lighter
Gel Electrophoresis
• Ethidium bromide causes
nicks in the DNA fragments
in response to UV light.
– The nicking of the DNA will
cause the DNA fragments to
migrate slower.
• Ethidium bromide is a
mutagen.
• Wear gloves when
handling anything which
contains or contained
ethidium bromide.
7