Gel electrophoresis

Gel electrophoresis
Allows to separate charged molecules in
electric field
Separates charged molecules by size using
molecular sieve

Gel electrophoresis
separation by charge
Separation in electric field:
The strength of electric field
Separation in buffer

Movement of charged particles to anode/cathode

Rate of migration
Rate of migration is dependent upon:
Electrical field strength
The stronger the current, the faster the movement

Net charge of particle

Viscosity of electrophoretic medium
In a liquid medium
In semisolid matrix

Conformation of molecule of interest

Linear or circular
Interaction with another molecules

Voltage
As the voltage applied to a gel is increased, larger
fragments migrate proportionally faster than small
fragments.
the best resolution of fragments larger than about 2 kb is
attained by applying no more than 5 volts per cm to the
gel
the cm value is the distance between the two electrodes, not the
length of the gel
http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.h
tml

Gel electrophoresis
separation by size
Molecular sieve
Is determined by the range of sizes need to be
separated

Creates certain size pores

Starch gels
Acrylamide gels
Agarose gels

DNA Migration on Agarose Gel

Because of an equal
charge to mass ratio
(one negative charge for
each nucleotide) the
DNA fragments are
separated based upon
their size and shape.
Linear DNA fragments
migrate different than
circular DNA

http://www.vivo.colostate
.edu/hbooks/genetics/bio
tech/gels/supercoils.jpg

Gel electrophoresis:
separation by charge
Buffers used:
Tris-EDTA-borate
Tris-EDTA-acetate
Stock prepared, 10X, diluted to 1X

Buffers
TAE is commonly used, easy to prepare and store
TAE should be used when DNA is to be purified from
the gel (TBE/agarose interaction will reduce the
amount of DNA purified)

TBE buffer has a superior buffering capacity

pKa of Tris (8.08) is closer to pKa of boric acid (9.24)
while acetic acid pKa is 4.76
Is not the best choice for large DNA fragments
Good for smaller sizes, interact with agarose more
tightly, effectively forming smaller pores
Current Protocols Essential Laboratory Techniques, 2nd edition,
Gallagher and Wiley

Gel Electrophoresis - equipment

Power supply
Gel box
Gel tray
Combs
Staining EtBr, ethidium bromide

Gel electrophoresis
separation by size
DNA is a large molecule
In order to separate on the gel, need to be
digested to smaller fragments
Naturally occurring in bacteria enzymes
restriction enzymes

Restriction Enzymes
Proteins that recognize specific sites in DNA
molecule
Restriction enzymes recognize 4-6 bp
palindromes
EcoRI GAATTC
CTTAAG

eplantscience.com

Discovery of a Bacterial Defensive

(Immune?) System
1950s host controlled restriction
1960s Werner Arber hypothesized
restriction/modification system
1970-72 Hamilton Smith purified the first
restriction enzyme and identified its
recognition sequence
1970s Dan Nathans used restriction
enzymes to construct restriction maps of SV40
viral DNA, Nobel Prize in 1978

Restriction/Modification Systems
Bacteriophage lambda and two E.coli strains
K12 and B
infects K12 isolate from plaques > infects
bacteria again
does not infect B strain of bacteria
but 0.01% - successful infection restricted infection.
isolate from plaques > will infect bacteria strain B
from strain B infection will not infect strain K12

Infect K12

E. coli K12

infect

http://sandwalk.blogspot.com/2007/05/
bacteriophage-lambda.html

Bacteriophage
lambda

Does not infect K12

E.coli B
Does not infect but 0.01%
successful infection
restricted infection

Infect B strain successfully

Restriction/Modification Systems
Different strains of E. coli possess different
restriction enzymes that recognize different
sites within a DNA molecule
Corresponding DNA methylation activity
R-M system restriction-modification
(methylation, methylases)

Restriction/Modification Systems
Phage that infected strain K12
same DNA modifications as bacterial DNA, restriction enzymes do not
recognize or cannot digest

The growth of the phage on strain B

not totally inhibited but effectively restricted

Phage that infected strain B

DNA of the phage has been modified during infection
K12 no specific modification of strain B to infect this strain.
Unmodified DNA vulnerable to attack by restriction
endonucleases of strain B

Restriction/Modification Systems
Methylation of adenine or cytosine residues inhibit
the action of a restriction enzyme
By modifying their genomic DNA by methylation,
bacteria prevents the restriction of their genetic
material with their restriction enzyme activity
Foreign DNA lacking the specific methylation pattern
is attacked and restricted by the restriction
endonuclease

http://2009.igem.org/Team:Imperial_College_London/M3/DamMethylation

Separation by size
How to determine the
size of a fragment?
Molecular ladders
Known size fragments
used as a reference
Bacteriophage digest
one of the first
Molecular Markers, BioRad

Molecular Markers determining the size of

DNA fragment
Run size standards in the gel
along with the samples of interest
A linear relationship is obtained
if the logarithms of the sizes (in
base-pair units) of the DNA
fragments are plotted against the
distance traveled through the gel
Construct a calibration curve for
distance run by the fragment of
certain size (d versus logM, Mmolecular weight, or length)
The size of the samples of
interest can be determined by
measuring the distance run d and
reading M from the calibration
curve.

http://www.bx.psu.edu/~ross/workmg/Struc_
Nucleic_Acids_Chpt2.htm

DNA migration in a gel

Calibration curve

Conformation of DNA

Gel picture and caption

Agarose gel electrophoresis of uncut and digested DNA. The gel was run at 120 V in Tris/borate/EDTA
Buffer for 1 hour, stained with ethidium bromide and viewed under UV light. Lanes 1 and 8 molecular
marker, lambda DNA digested with EcoRI. Lanes 2 vector undigested, lane 3 vector digested with EcoRI.
Lanes 4 7: recombinant vectors A or B undigested, digested with EcoRI or HindIII/SalI
http://homepages.strath.ac.uk/~dfs99109/BB211/ResMap.html

Determine the size of the fragment

23.7 kb - 0.75 cm
9.46 kb 1.35 cm
6.75 kb 1.85 cm
4.26 kb 2.6 cm
3.1 cm- kb?
2.26kb 4.05 cm
1.98kb 5.3 cm

http://www.mun.ca/biology/scarr/Gel_Electrophoresis.html

Practical use restriction mapping

One of the first applications restriction maps
of plasmids

http://www.bio.miami.edu/~cmallery/150/gen
e/c7.20.8.electrophoresis.jpg

Lambda DNA
http://www.apsnet.org/educatio
n/k-12plan

Restriction Digest of pSR4380

Restriction Digest of pSR4380

E

E/H

H/B

E/B

E/B/H
6
5
4

3
2

1.5

0.5
6 kb

6 kb

1kb
5 kb

Restriction Digest of pSR4380

EcoRI
0/6 kb

Mapping Guidelines
Label Lanes
Determine how many times each enzyme cuts plasmid
Add up fragment sizes of each lane
(Are some bands doublets?)

Determine total size of a plasmid

Start a map pick 12 oclock position hold constant!
Compare double digests that use the 12 oclock reference
Compare double digests that do not use the 12 oclock
reference
Compare triple digest

Experiment planning:
questions to ask yourself before running a gel

What do you want to separate on the gel?

What size fragments do you expect?
How much DNA should you use?
What size of the gel do you need?
What percent agarose do you need?
What ladder will you use?
How will you stain DNA?

The Week in Review

Experiment 2
Gel Electrophoresis of Pre-digested DNA

Pour a 1% agarose gel (in pairs).

Watch demonstration of loading samples on a gel.
Practice loading gel.

You will separate fragments of pre-digested plasmid

DNA samples and begin electrophoresis.
Take a picture of agarose gel results
Use the picture to re-create the plasmid map

Be able tos
Be able to describe the scientific basis behind the
separation and visualization of DNA on agarose
gel
Be able to create a calibration curve for DNA
separation and determine a size of a fragment on
a gel
Be able to draw a plasmid map based on the DNA
pattern on agarose gel.
Be able to draw a DNA pattern on agarose gel
given a plasmid map and various restriction
digest conditions