Download Agarose Gel Electrophoresis

AGAROSE GEL ELECTROPHORESIS
IMBB 2016
BecA-ILRI Hub, Nairobi
May 9 – 20, 2016
Martina Kyalo
Terms
• Gel electrophoresis is a method that uses an
electrical current and a gel matrix to separate
molecules like DNA and proteins.
• Buffer a solution containing either a weak
acid and its salt or a weak base and its salt,
which is resistant to changes in pH.
Agarose Gel Electrophoresis
This is a procedure that separates molecules
on the basis of their rate of movement through
agarose gel under the influence of an electrical
field.
• Separates DNA or RNA by:
 size and/or
 charge and/or
 shape
Reagents and Supplies
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Weighing scale
Spatula
Flask
Graduated cylinder
Microwave
Agarose
Buffer
Gel tray(s) and comb(s)
Gel box(es)
Power supply
DNA Staining solution
Photo doc. system
Electrophoresis Equipment
Power supply
Cover
Gel tank
Electrical leads

Casting tray
Gel combs
Agarose
• Agarose is a linear polymer extracted from
seaweed.
• An agarose gel is used to slow the
movement of DNA .
 Within an agarose gel, linear DNA
migrate inversely proportional to their
molecular weight.
Resolution of linear DNA
fragments in agarose gel
% Agarose (w/v) Size Range (kb)
for Optimal Separation
0.5
0.75
1.0
1.5
2.0
2 - 30
0.7 - 20
0.5 - 10
0.2 - 3
0.1 - 2
Buffer Systems
• Weak acids and/or bases that do not dissociate
completely.
• Purposes of buffer:
– Maintain pH.
– Generate ions consistently to maintain current &
keep resistance low.
1)TAE, pH 8.0, ~50 mM - Tris, Acetate, EDTA
2)TBE, pH 8.0, ~50 mM - Tris, Borate, EDTA


•
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TBE resolves low MW fragments better than TAE.
TAE resolves high MW fragments better than TBE
Tris (T) is a weak base.
Acetic (A) acid & boric (B) acid are weak acids.
Pouring a horizontal agarose gel
Visualization
Monitoring the progress of the electrophoresis
• Tracking dyes are visible to naked eye during run
→Xylene cyanol (migrates with ~5.0 kb fragments)
→Bromophenol blue (migrates with 300 bp fragments)
→Orange G (migrates with fragments of ~50 bp)
But
• Mobility of tracking dyes can vary substantially
depending on agarose
→Concentration
→Type
DNA stain
• Binds to ds DNA by intercalation between
stacked bases.
• Used to visualize DNA with UV light.
– E.g. Ethidium bromide, GelRed
***CAUTION!
– UV light damages eyes and skin! Wear goggles and/or face
shield.
– Ethidium bromide is a powerful mutagen and is moderately
toxic. Gloves should be worn at all times.
How fast will the DNA migrate?
 Strength of the electrical field
 Size of the DNA
 Buffer
 Concentration of agarose gel used
DNA
small
large
-
Power
+
What factors affect mobility of
linear ds DNA?
• Pore size of the gel
–  [agarose]   pore size
–  pore size   friction   mobility
• Voltage across the gel
–  voltage   mobility
• Length of the DNA molecule
– smaller molecules generate less friction
and so move faster
Factors affecting resolution
Resolution = separation of fragments
The “higher” the resolution, the more space
between fragments of similar, but different,
lengths.
Resolution is affected by
agarose concentration
salt concentration of buffer or sample
amount of loaded DNA
voltage
Why run an agarose gel?
• Determine the quality or quantity of DNA
• Estimate the size of DNA molecules
• Purification of DNA
• Analyze PCR products
– Molecular diagnosis or genotyping
Genomic DNA
M
1
2
3
M = 1kb + DNA ruler
1 = Lambda DNA (control)
2 - 3 = gDNA
PCR products
M
M
3a
4a 4b 1a
= 1kb + DNA ruler
1b
2a
Msel digestion of PCR products
M
= 1kb + DNA ruler
Genomic DNA
1
= Lambda DNA (control)
2 – 13 = gDNA
PCR products
M
= 1kb + DNA ruler
Trouble shooting
• Smearing
– torn sample wells
– voltage too high for large fragments
– too much DNA
• Gel melts
– voltage too high
– ionic strength too low
• Poor resolution
– wrong agarose concentration
– small bands are fuzzy –diffusion of the DNA
and broadening of the band