6. Gel
electrophoresis
A gel is a colloid, a
suspension
of
tiny
particles in a medium,
occurring in a solid form
(like gelatin).
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7. Gel electrophoresis refers to
the separation of charged
particles located in a gel
when an electric current is
applied.
Charged
particles
can
include DNA, amino acids,
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polypeptides, etc.
7
12. Agarose
Larger 'pores'
Large particles can
move more easily
Used for the
electrophoresis of large
molecules such as DNA
and RNA
Agarose gels have a
large range of
separation, but relatively
low resolving power
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Polyacrylamide
Small pores
Large particles cannot
move easily
Used for
electrophoresis of small
molecules such as
proteins
Polyacrylamide gels
have a rather small
range of separation, but
very high resolving
power
14. The agarose gel after solidification has
some gaps called PORES.
These pores are the channels through
which charged molecule has to travel.
When charged molecules are placed in an
electric field, they migrate toward either
the positive or negative pole according to
their charge.
Nucleic acids have negative charge due to
their phosphate backbone.
The migration flow is determined solely
by the molecular weight.
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small weight molecules migrate faster
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than larger ones.
18. Materials required
DNA sample
Agarose
Ethidium bromide
6X sample loading dye
DNA ladder standard
Power supply
Gel casting tray &
comb
Pipette and tips
Electrophoresis
chamber
Buffers
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Transilluminator
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The movement of
suspended particles through
a medium under the
action of an electromotive
force applied to
electrodes in contact
with the
suspension”
21. 3. Pour the gel in
casting tray
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22. 4. Insert the comb and let the
gel solidify
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23. 5. After solidification of gel, carefully
remove the comb and place the gel in
the gel rig with the wells closest to
the cathode (black) end. Cover the
gel with 1X TAE running buffer.
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24. 6. Keeping DNA samples on
ice, pipette up 5 µl of a
sample, add it to one drop
(1μl) of loading dye.
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25. 7. Load the mixture into
a well
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26. 8. Place the cover on the gel rig
and run the samples towards the
anode (red) end. And run it at
120 volts for about half an hour.
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27. 9. Turn off the power pack,
remove the gel and visualize
with transilluminator or U.V.
light.
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28. 10. Take a photograph with a
polaroid Photo documentation
camera.
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30. Ethidium bromide
is used for staining
It will bind with DNA and & it will
emitt fluoresence
mutagenic and should be handled
with extreme caution
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31. Loading dye
Gives colour and density to the
sample
the dyes are negatively charged
and thus move in the same
direction as the DNA
Bromophenol blue for dying
Glycerol for density
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32. DNA ladder
standard
useful for quantifying
the amount of DNA in
your sample bands by
comparison with the
marker bands
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33. Tank buffers
TAE buffer (Tris Acetate EDTA)
TBE buffer (Tris borate buffer)
sequesters divalent cations
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34. TAE buffer
TAE offers the best resolution
for larger DNA
TAE requires a lower voltage
and more time
linear, double stranded DNA
runs faster in TAE.
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35. 1X TAE Buffer:
1. Trisma base = 242g
2. Glacial acetic acid =
57.1ml
3. 0.5M EDTA = 100ml
Mix all the constituents and
raise the
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volume up to 1L with distilled
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40. Unique bands and or
banding patterns.
Molecular weight standards
can be ran with samples to
estimate sizes..
Migration or separation
of a sample can
be measured (Rf values).
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44. It can be used to
determine
the
sequence
of
nitrogen bases.
the size of an insertion or deletion
the presence of a point mutation
to distinguish between variable
sized alleles at a single locus
to assess the quality and quantity
of DNA present in a sample
DNA Sequencing
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45. Blotting (Southern Blot)
Size of DNA can be determined by
comparing it with ladder.
Medical Research
Forensics (Criminal investigations)
like Blood, Saliva, Hair, nail
Paternity Tests
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48. Disadvantage
Toxicity
DNA visualization uses dyes like
Ethidium Bromide which chelate DNA
and is a known carcinogen. Although
some safer alternatives are becoming
available (SYBR Safe).
•
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50. Definition
A technique which allows
the detection of a specific
DNA sequence (gene
or
other) in a large, complex
sample of DNA.
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51. Principle
Southern blotting combines
agarose gel electrophoresis
for size separation of DNA
with methods to transfer the
size separated DNA to a
filter membrane for probe
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hybridization.
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52. Procedure
Digestion + gel electrophoresis
DNA is transferred to a sheet
of special blotting paper
Label with specific DNA probe
Detected on the Xray film
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54. Applications
To identify a single gene among
thousands of fragments of DNA and
to detect sequences of DNA in an
organism’s genome.
Used in gene discovery and gene
mapping.
To analyze the genetic patterns in an
organism’s DNA.
To identify gene
mutations, deletions, duplications, a
nd gene rearrangements involved in
diseases.
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To determine the number of copies
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55. To identify related DNA sequences in
the genome and to determine if there is
a gene family (a group of similar genes.
To detect certain cancers and genetic
diseases, such as:
Monoclonal leukemia populations
Sickle cell mutations
Used in DNA fingerprinting, genetic
engineering, & forensic science for
tests such as
Paternity testing
Sex determination
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