Transcript Agarose Gel Electrophoresis - Cal State LA

Agarose Gel
Electrophoresis
What does gel electrophoresis do?
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employs electromotive force to move molecules
through a porous gel
separates molecules from each other on the
basis of
 size
and/or
 charge and/or
 shape

basis of separation depends on how the sample
and gel are prepared
Why perform electrophoresis on
ds DNA?
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To separate fragments from each other
To determine the sizes of fragments
To determine the presence or amount of DNA
To analyze restriction digestion products
Length
standards
Ethidium bromide stained gel photographed
on UV light box in black and white
power
supply
gel box
Pouring a horizontal
agarose gel
Where does the current come from?
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A direct current power supply
Ions supplied by the buffer
The charge on the macromolecules being
separated
Electrolysis of water
Where does the current come from?
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Electrolysis of water
 4H2O
 2H2 + O2 + 2H2O
 self-ionization
of water throughout the buffer:
4H20  4H+ + 4OH At
the negative pole
• 4H+ + 4e-  2H2
 At
the positive pole
• 4OH-  O2 + 2H2O + 4e At
which electrode would you expect more
bubbles? Why?
Basics of Gel Electric Circuits
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V (volts) = I (milliamps) X R (resistance)
For a segment of a gel/buffer system

cross-sectional area of buffer or gel,  resistance
  strength of buffer = [ion],  resistance
 most resistance is in the agarose gel itself
What factors affect mobility of linear
ds DNA?
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Pore size of the gel

[agarose]   pore size
  pore size   friction   mobility
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Voltage across the gel

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voltage   mobility
Length of the DNA molecule
 smaller
molecules generate less friction and so
move faster

Ethidium bromide (stain) intercalated into DNA
 decreases
mobility
charge to mass ratio and so decreases
Why don’t charge and shape affect
mobility of linear ds DNA?

all DNA molecules have an essentially identical
charge to mass ratio
1
negative charge/phosphate and 1phosphate/base,
so . . . .
 charge is directly proportional to length
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different lengths have essentially identical rod
shape
Note: shape does affect mobility of circular and/or
single strand DNA or RNA
Visualization

Monitoring the progress of the electrophoresis
 tracking
dyes visible to naked eye during run
 xylene
cyanol (migrates with ~5.0 kb fragments)
 bromphenol blue (migrates with fragments of a few
hundred base pairs)
 Orange G (migrates with fragments of ~50 bp)
 but
mobility of tracking dyes can vary substantially
depending on agarose
 concentration
 type
Visualization
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Locating the DNA fragments in the gel
 ethidium
bromide staining
 mutagen,
wear gloves!
 visible under UV light
 wear UV opaque face or eye shield to observe!

Locating the DNA fragments in the gel
 comparison
to length standards
Length
standards
Ethidium bromide stained gel
photographed on UV light box in black
and white
Factors affecting resolution
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Resolution = separation of fragments
The “higher” the resolution, the more space between
fragments of similar, but different, lengths
Resolution is affected by
 agarose
type
 agarose concentration
 salt concentration of buffer or sample
 amount of DNA loaded in the sample
 voltage
What is agarose?
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linear carbohydrate polymer extracted from
seaweed , agarbiose
forms a porous matrix as it gels
 shifts
from random coil in solution to structure in
which chains are bundled into double helices
What is agarose? (cont’d)
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multiple types of agarose
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Standard agarose - LE
Gels at 35-38oC; Melts at 90-95oC
 Becomes opaque at high concentrations
 Makes a fairly sturdy gel
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Low melting agarose (NuSieve)
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Gels at 35oC; Melts at 65oC
• Often used to isolate DNA fragments from gel
Modified by hydroxyethylation to lower M.P.
 Is relatively translucent at high concentrations
 Makes a fragile gel
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Intermediate forms or combinations of LE and NuSieve can
provide sturdy, translucent gels at high agarose
concentrations
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Good for resolving smaller fragments
Resolution of ds linear DNA fragments in agarose gels
% Agarose (w/v) Size Range (kb prs)
for Optimal Separation
0.5
0.75
1.0
1.5
2.0
3.0 (Nu-Sieve)
4.0 (N-S)
5.0 (N-S)
6.0 (N-S)
2-30
0.7-20
0.5-10
0.2-3
0.1-2
0.07-1.5
0.04-0.9
0.03-0.6
0.01-0.4
0.7%
2.5%
Effect of agarose concentration on linear DNA fragment resolution.
The two lanes contain identical DNA samples.
4M
1M
0M
Effect of salt concentration on resolution of fragments.
Samples in all three lanes are identical except for [salt].
0.1
0.25
0.5
1.0
5.0
 DNA Hind III fragments (g)
Effect of sample DNA concentration on resolution.
Voltage
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 voltage,  rate of migration
to increase the voltage
 increase
the setting on the power supply
 increase the resistance
 decrease
the gel thickness
 decrease the ion concentration
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if voltage is too high, gel melts
as voltage is increased, large molecules migrate at a
rate proportionally faster than small molecules, so
 lower
voltages are better for resolving large fragments
 but the larger ds DNA fragments are always slower
than the smaller ones
Buffer Systems
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Remember, buffer systems include weak acids
and/or bases that do not dissociate completely.
 If
ions resulting from dissociation are “removed,”
more weak acid and/or base will dissociate.
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Purposes of buffer
 Keep
solution at pH compatible with molecules
being separated
 Generate ions consistently to
 maintain
current
 keep resistance low
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Both gel and the solution in the gel box are
buffered.
Buffer Systems (cont’d)
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Two commonly used buffers for routine agarose gel
electrophoresis
TAE, pH 8.0, ~50 mM - Tris, Acetate, EDTA
 TBE, pH 8.0, ~50 mM - Tris, Borate, EDTA
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Tris (T) is a weak base.
Acetic (A) acid and boric (B) acid are weak acids.
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Acetic acid is more completely ionized at pH 8.0 than is
boric acid, so TBE has a high buffer capacity than TAE.
Buffer Systems (cont’d)
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TAE, pH 8.0, ~50 mM - Tris, Acetate, EDTA

loses buffer capacity during long or high voltage gel runs;
• anode end of gel becomes acidic
• gel may melt from the increased resistance that results from
ion depletion
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resolves high MW fragments better than TBE
TBE, pH 8.0, ~50 mM - Tris, Borate, EDTA
higher buffer capacity
 somewhat more expensive
 resolves low MW fragments better than TAE
 may interfere with subsequent reactions
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Non-denaturing agarose gel loading
solutions
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Composition
 tracking
dyes
 are
used to follow progress of electrophoresis
 sometimes interfere with later visualization of DNA
a
solute to increase density
 so
that sample falls to bottom of loading well with
minimal dilution
 solute examples: glycerol, Ficoll
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Other gel types, with different purposes, use
different loading solutions!
Ethidium bromide staining
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Binds to DNA by intercalation between stacked bases
lies perpendicular to helical axis
 makes Van der Waals contacts with bases above and below
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+ charge,
so migrates toward negative pole
 reduces the charge to mass ratio of the DNA fragment to which
it is bound
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Because of the change in charge to mass ratio, it alters DNA
mobility, especially of circular covalently closed DNA
Ethidium bromide staining
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Used to visualize DNA with UV light
 254 nm absorbed by DNA and transmitted to EtBr
 excitation at 302 or 366 nm  fluorescence at 590 nm
 uv
 560
 >/=
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nm = visible red/orange
10ng/band required for visualization
Bound dye fluoresces 20-25X more than dye in
solution because of
 fixed
position of planar group
 proximity of dye to bases
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UV light damages eyes and skin! Wear goggles
and/or face shield.
Trouble shooting
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Smearing
torn sample wells
 voltage too high for large fragments
 too much DNA
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Use </= 0.5 ug / fragment / 0.25cm2 migration area
Gel melts
voltage too high
 ionic strength too low
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Poor resolution
wrong [agarose]
 small bands are fuzzy – the gel run may have been too long at
too low a voltage, allowing diffusion of the DNA and
broadening of the band
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