Transcript DNA GEL ELECTROPHORESIS

4-4 GEL ELECTROPHORESIS:
Using Gel Technology to Study DNA
Molecules
What is Gel Electrophoresis?
• Electro- = flow of electricity
• -phoresis = to carry across
• A gel is a suspension of tiny particles in a
medium, occurring in a solid form (like
gelatin)
• Gel electrophoresis = a process that
uses electricity to separate charged
molecules- DNA, RNA, and proteins, on a
gel slab.
Examples
• Gel Electrophoresis is
used to visualize…
– Genomic DNA
– RNA
– Polypeptides
– PCR products
– Plasmids
– Restriction enzyme
digest products
http://www.steve.gb.com/images/science/agarose.jpg
Two Types of Gels
1) Agarose gels
 Medium to Large DNA fragments or RNA
 Made from polysaccharides found in various
marine species of red algae (Agar)—
Some found right here in California!
Agar is composed of both agarose and
agaropectin molecules and provides support to
the cell walls within the marine algae. Removed
from the plant, the agar can be used as a food
thickener, much like gelatin, a laxative, or a
medium for growing bacteria, fungus, or other
microorganisms, when purified.
Two Types of Gels
1) Agarose gels
 The Agarose is purified and dissolved in a
boiling buffer solution (TAE)
 When solidifies it provides network of pores (a
matrix) for particle movement
 Run in horizontal gel boxes
 Gel concentration depends on the size of the
DNA fragment of interest
 Most common stain to visualize the DNA:
Ethidium bromide (EtBr) or Methylene blue
Two Types of Gels
2) Polyacrylamide Gels (PAGE)
 For proteins and very small DNA/RNA
fragments (get finer resolution of bands)
 Made from the polymer, polyacrylamide (toxic
in liquid state; non-toxic in solid state)
 Run in vertical gel boxes
 Gel concentration depends on the size of the
protein of interest
 Most common stain to visualize Proteins:
Coomassie Blue or Silver Stain
Why Perfom gel electrophoresis?
• When DNA is cut by restriction enzymes*,
the result is a mixture of DNA segments of
varying lengths
• It is useful to be able to separate the pieces
(for forensic work or for DNA sequencing)
*Reminder: …. A few words about
restriction enzymes!
History of Restriction Enzymes
 Restriction enzymes are called restriction
endonucleases- cut DNA strand at certain
nucleotide sequences usually 4-6 base pairs
long
 Occur naturally in some species of bacteria
where their role is “defense”
 These enzymes “restrict” foreign (e.g. viral)
DNA that enters the cell, by destroying it—
cutting the invader DNA at certain
segments.
 The host cell DNA is protected
 ~800 known restriction
enzymes
 1971- Biotechnology uses
restriction enzymes
 Restriction Digests – put
restriction enzyme with DNA of
interest to get known DNA
fragments
 Restriction Enzymes/Uses:
Lambda DNA (virus that
infects bacteria) cut with
HindIII—most common
molecular weight marker
to run as standard on
gels
EcoRI (enzyme from E.
coli)
BamHI (from Bacillus
amyloliquefaciens)
How does gel
electrophoresis work?
• DNA is an organic macromolecule that has an
overall negative charge due to the negative
charge on the phosphate groups of the
backbone
• When the DNA is exposed to an electrical
field, the particles migrate toward the
positive electrode (hint: think “run red”)
How does it work?
• The solution is poured into gel trays with
plastic combs. These combs help mold the
wells when the hot liquid is poured into the
gel tray. When the gel solidifies, wells are left
which provide a place to load your DNA.
• The solidified gel is placed into a gel box
• The gel is covered with TAE buffer and the gel
box is filled with the TAE buffer
How does it work?
• When the power supply is turned on, an
electric current runs into the gel box, and an
electric field is established between the
positive and negative electrodes.
• The charged molecules move out of the wells
and into the gel. If the molecule has a
negative charge, they migrate towards the
positive electrode (DNA). If the molecule has
a positive charge, they move toward the
negative electrode.
How does it work?
• DNA fragments migrate toward the positive
electrode at a rate that depends on their size
and the size of the pores in the gel.
– Large DNA fragments= move slower and stay
closer to the wells
– Smaller DNA fragments = move faster and move
farther from the wells
Different gel []
• The concentration of the gel (matrix density) is
critical
• The more agarose = the more compact and
tighter the matrix = harder for charged particles
to move through the gel
• Most agarose gels are made between 0.6% and
3%.
– A 0.8% gel (most common) will show good
resolution (separation) of large DNA
fragments (500-10,000 bp)
Different gel []
– A 2%-3% gel will show good resolution for
small DNA fragments (200-1,000 bp)
• Lower percentage gels (0.6%) are very weak and
may break when you try to lift them. They
separate only VERY Large DNA = genomic
DNA.
• High percentage gels are often brittle and do
not set evenly.
What is needed?
• Solidified
Agarose gel
• Power
supply
• Gel box
(chamber)
What is needed?
• Buffer - either TBE or TAE
–The buffer provides ions in solution
to ensure electrical conductivity
• Contains TRIS, a buffering salt that stabilizes
the pH and helps maintain the shape of the
molecules being analyzed
– Not only is the agarose dissolved in buffer,
but the gel slab is submerged in the buffer
after adding it to the gel box
What is needed?
• Sample Loading Buffer –
– To visualize sample movement through the
gel (you ARE NOT visualizing the DNA as
it moves through the gel!)
• We cannot “see” the DNA until we stain it
– Helps “sink” the DNA into the wells (glycerol or
equivalent)
– The loading dye helps us keep track of how far the
DNA has migrated through the gel
• You don’t want your DNA to run off the gel!
What is needed?
• Molecular weight
markers (Ladders)DNA fragments of known
size (Lambda/HindIII)
• Comparison of your sample
bands to the marker bands
allows…
– Visible confirmation of
desired product
– Quantification of sample
DNA
http://www.biomedcentral.com/content/figures/1471-2180-5-63-2.jpg
Gel electrophoresis
- electrode
DNA fragments
+ electrode
Agarose gel
~~~~~~~~~~~~~~~~~~~~~~~~ buffer ~~~~~~~~~~~~~~~~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~~~
Gel electrophoresis
- electrode
+ electrode
current
~~~~~~~~~~~~~~~~~~~~~~~~ buffer ~~~~~~~~~~~~~~~~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~~~
Visualizing DNA
• Most of the time Ethidium bromide
(EtBr) is used
–A fluorescent dye visualized when
excited by UV light
–Intercalates (inserts) into the DNA
molecule, thereby “staining” it
Visualizing DNA
• Gel is soaked in a solution of EtBr and
the DNA bands take up the dye (or
EtBr is added directly to the gel
solution during preparation)
• Then the gel is then placed on a light
box, exposed to UV light, and a
photograph is taken for
documentation
EtBr Gel Under UV Light
Brighter bands= high concentration of DNA
Lighter bands = low concentration of DNA
**Each band that you see is a collection of millions of
DNA molecules, all of the same length!!
Visualizing DNA
• Ethidium Bromide is a mutagen
• So we will use trace amounts of EtBr
in our gels to minimize student
exposure to the compound
• Listen to instructions when
pouring your gels or analyzing your
gels! (CMB)
Important!
• A gel only shows us the relative size
of the DNA fragments. It DOES NOT
tell us the sequence of those
fragments
– DNA sequencing is the next step to
determining the actual nucleotide
sequence of the DNA fragment(s).
Gel Electrophoresis
Prepare agarose gel
Melt, cool and add Ethidium Bromide. Mix thoroughly.
Pour into casting tray with comb and allow to solidify
Add running buffer, load samples and marker
Run gel at constant voltage until band separation occurs
View DNA on UV light box and document results
Helpful Hints
• When placing the gel in the electrophoresis
chamber:
– Make sure that the wells are closest to the
negative (black) electrode (since DNA is
negative)
• When adding the buffer to the chamber:
– Gently flood the gel from the end opposite the
wells to minimize sample diffusion
• Before loading the wells:
– Orient the entire chamber close to the power
supply so it is in place when the samples are
ready to run
Helpful Hints
• When loading samples in the wells of the gel:
– Use proper micropipette techniques
– Make a written record of which sample you
will load in each well of the gel
– Be careful not to puncture the bottoms of the
wells as you load the samples
– If you make a mistake, do NOT take more than
the allotted sample; there are limited amounts
Troubleshooting Guide
DNA Science: A First Course in Recombinant DNA Technology, by David A. Micklos and Greg A. Freyer
Gel electrophoresis separates
molecular products based
on:
A. Shape
B. Size
C. Charge
D. Evolutionary similarity
Why does DNA move through
a gel?
1. The negative charge of DNA is attracted to the
positive end of the electrophoresis box.
2. The negative charge of DNA is attracted to the
negative end of the electrophoresis box.
3. The positive charge of DNA is attracted to the
positive end of the electrophoresis box.
4. The positive charge of DNA is attracted to the
negative end of the electrophoresis box.
What is the purpose of
loading dye in gel
electrophoresis?
1. To see the DNA in the gel
2. To help your sample sink into the
well
3. To “see” movement on the gel so
you can turn it off before any DNA
runs off the edge of the gel
4. Both 2 & 3
What is the purpose of a DNA
ladder?
1. So you can estimate the length of
DNA fragments running through a
gel
2. It’s a control for your experiment
3. It helps DNA samples move through
the gel
4. Both 1 & 3
When traveling through an agarose gel,
larger molecular products will
migrate _________ smaller
molecular products.
A. Faster than
B. Slower than
C. At the same rate
Which fragment is the largest?
1.
2.
3.
4.
Fragment
Fragment
Fragment
Fragment
#1
#2
#3
#4
1
2
3
4
The size of the molecular
products is determined by:
A. The intensity of the band
B. The percentage of agarose gel
C. Comparison with a molecular weight
“ladder”
D. Being familiar with your gel and
“eyeballing” it
The concentration of the
molecular products is
determined by:
A. The intensity of the band
B. The percentage of agarose gel
C. Comparison with a molecular weight
“ladder”
D. Being familiar with your gel and
“eyeballing” it
Buffer is used instead of water
when making and running
gels because:
A. Buffer enhances the transmission of
electric currents in water
B. Buffer is more homogeneous than
water
C. Buffers provide the salts necessary
to help create the current
How Gel Electrophoresis
Works
http://www.sumanasinc.com/webcontent/anisamples/majorsbiology/
gelelectrophoresis.html
http://learn.genetics.utah.edu/units/biotech/gel
If this is a P200, what
volume will be measured?
1.
2.
3.
4.
1000uL
100uL
10uL
1uL
If this is a P1000, what
0
volume will be measured?
1.
2.
3.
4.
3000uL
300uL
30uL
3uL
3
0
If this is a P20, what volume
will be measured? 1
1.
2.
3.
4.
1730uL
173uL
17.3uL
1.73uL
7
3
What is the purpose of the
1st stop on a micropipettor?
1.
2.
3.
4.
Measure correct volume
Eject entire volume from the tip
Eject the tip from the pipette
None of the above
Is this centrifuge
balanced?
1. yes
2. no