Polyacrylamide Gel Electrophoresis

2. Introduction of Gel Electrophoresis
Why do gel electrophoresis
 Electrophoresis Equipment
How does it work?
Steps Of Gel Electrophoresis
Identification Of Novel Mutation By Gel
Electrophoresis
2
Contents

3.  Electro = flow of electricity, phoresis, from the
Greek = to carry across
 A gel is a colloid, a suspension of tiny particles in
a medium, occurring in a solid form, like gelatin
 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,
peptides, etc

4.  When DNA is cut by restriction enzymes, the
result is a mix of pieces of DNA of different
lengths
 It is useful to be able to separate the pieces - I.e.
for recovering particular pieces of DNA, for
forensic work or for sequencing

5.  Polyacrylamide
 - Polyacrylamide is
dissolved in buffer and
heated, then cools to a
gelatinous solid with a
network of crosslinked
molecules
 Some gels are made
with agraose if lower
bands are required

6.  Buffer - in this case TBE
 The buffer provides ions
in solution to ensure
electrical conductivity.
 Not only is the
Polyacrylamide
dissolved in buffer, but
the gel slab is
submerged (submarine
gel) in buffer after
hardening

7.  Also needed are a
power supply and a
gel chamber
 Gel chambers come
in a variety of
models, from
commercial through
home-made, and a
variety of sizes

8. Casting tray
Gel combs
Power supply
Gel tank Cover
Electrical leads

Electrophoresis Equipment

9. Gel casting tray & combs

10.  DNA is an organic acid, and is negatively
charged (remember, DNA for Negative)
 When the DNA is exposed to an electrical field,
the particles migrate toward the positive
electrode
 Smaller pieces of DNA can travel further in a
given time than larger pieces

11. Seal the edges of the casting tray and put in the combs. Place the casting tray on a
level surface. None of the gel combs should be touching the surface of the casting
tray.
Preparing the Casting Tray

12. Each of the gel combs should be submerged in the melted Polyacrylamide solution.

13. When cooled, the Polyacrylamide polymerizes, forming a flexible gel. It should
appear lighter in color when completely cooled (30-45 minutes). Carefully
remove the combs and tape.

14. Place the gel in the electrophoresis chamber.

15. buffer 
Add enough electrophoresis buffer to cover the gel to a depth of at least
1 mm. Make sure each well is filled with buffer.
Cathode
(negative)
Anode
(positive)

wells
  
DNA

16.  DNA is prepared by digestion with restriction
enzymes
 Polyacrylamide is made to an appropriate
thickness (the higher the % Polyacrylamide,
the slower the big fragments run) and ‘melted’
in the microwave
 The gel chamber is set up, the ‘comb’ is
inserted
 The Polyacrylamide may have a DNA ‘dye’
added (or it may be stained later). The
Polyacrylamide is poured onto the gel block
and cooled

17.  The comb is
removed, leaving
little ‘wells’ and
buffer is poured over
the gel to cover it
completely
 The DNA samples
are mixed with a
dense loading dye
so they sink into their
wells and can be
seen

18.  The DNA samples
are put in the wells
with a micropipette.
 Micropipettes have
disposable tips and
can accurately
measure
1/1,000,000 of a litre

19.  The power source is turned on and the gel is run.
The time of the run depends upon the amount of
current and % gel, and requires experimentation
 At the end of the run the gel is removed (it is actually
quite stiff)
 The gel is then visualized - UV light causes the
bands of DNA to fluoresce

20. Place the cover on the electrophoresis chamber, connecting the electrical leads.
Connect the electrical leads to the power supply. Be sure the leads are attached
correctly - DNA migrates toward the anode (red). When the power is turned on,
bubbles should form on the electrodes in the electrophoresis chamber.
Running the Gel

21.  wells
 Bromophenol Blue
Cathode
(-)
Anode
(+)
Gel
After the current is applied, make sure the Gel is running in the correct
direction. Bromophenol blue will run in the same direction as the DNA.
DNA
(-)


22. A gel as seen under UV light - some samples had 2 fragments
of DNA, while others had none or one

23. Visualizing the DNA (QuikVIEW stain)
 250
 1,500
 1,000
 500
 750
 2,000 bp
DNA ladder

PCR
Product
wells
+ - - - - + + - - + - +
March 12, 2006
Samples # 1, 6, 7, 10 & 12 were positive for Wolbachia DNA

24.  Many samples can
be run on one gel-
but it is important to
keep track
 Most gels have one
lane as a ‘DNA
ladder’ - DNA
fragments of known
size are used for
comparison

25.  The DNA band of interest can be cut out of the gel
and the DNA extracted -
 Or DNA can be removed from the gel by Southern
Blotting

26.  Compare the two sheets of electrophoresis of the
same animals.
 If any mutation occur in the animal than the band
shows the different position in electrophoresis sheet.
 the DNA ladder shows how much nucleotides are
differs form normal and mutant.

27.  www.biotech.iastate.edu
 Biotechnology by G. Mischer
 Amoeba sister (www.youtube/amoebasister.com)
 Tolls of Genetics
 Photos - L. D. Macdonald, 2003

28.  Patricia Barril and Silvia Nates (2012). Introduction to
Agarose and Polyacrylamide Gel Electrophoresis
 Matrices with Respect to Their Detection Sensitivities,
Gel Electrophoresis - Principles and Basics, Dr. Sameh
 Magdeldin (Ed.), ISBN: 978-953-51-0458-2, InTech,
Available from:
http://www.intechopen.com/books/gelelectrophoresis-
 principles-and-basics/introduction-to-agarose-and-
polyacrylamide-gel-electrophoresismatrices-
with-respect-to-their-detect