includes basics of gel electrohoresis

1. GEL ELECTROPHORESIS

2. Why gel electrophoresis?
•To separate DNA fragments from each other
•To determine the sizes of DNA fragments
•To separate RNA and Proteins.

3. Gel electrophoresis is a widely used technique for the analysis of
nucleic acids and proteins.
Gel electrophoresis separates molecules on the basis of their rate
of movement through a gel under the influence of an electrical
field.
Based upon medium of gel electrophoresis-
AGE (Agarose gel electrophoresis)
PAGE (Polyacrylamide gel electrophoresis)

4. Key components of Gel Electrophoresis
Gel type & concentration
Buffer System
Voltage/Current
Visualization

5. Agarose gel electrophoresis
(AGE)
Agarose is a natural linear polymer extracted from seaweed that
forms a gel matrix by hydrogen-bonding when heated in a buffer.
- low resolving power

6. Polyacrylamide gel electrophoresis
(PAGE)
Polyacrylamide gels are chemically cross-linked gels formed by the
polymerization of acrylamide with a cross-linking agent N, N’-
methylenebisacrylamide by free radical polymerization.
Greater resolving power
Neurotoxin (when unpolymerized)

7. Agarose
D-galactose 3,6-anhydro L-galactose
Polyacrylamide

8. Gel concentration
Agarose gel: In the range of 0.2% to 3% w/v.
The lower the concentration of agarose, the faster the DNA
fragments migrate.
In general, if the aim is to separate small DNA fragments, a high
concentration of agarose should be used.

9. Polyacrylamide gel: Depend upon ratio of acrylamide and the
cross linking agent, methylenebisacrylamide.
Researchers have settled on 5% (19:1 acrylamide/ bisacrylamide) for
most forms of denaturing DNA and RNA electrophoresis, and 3.3%
(29:1) for most proteins, native DNA and RNA gels.

10. Buffer system
• Buffers are an integral part of any electrophoresis technique.
• Electrophoretic mobility of DNA is affected by the ionic strength
(salt content) of the electrophoresis buffer.
• Without salt, electrical conductance is minimal and DNA barely
moves. In a buffer of high ionic strength, electrical conductance is
very efficient.
Dissociating and non-dissociating
Continuous and discontinuous.

11. Tris-Acetate-EDTA (TAE)
Tris-Borate-EDTA (TBE)
*at a concentration of approximately 50mM (pH 7.5-7.8).
• TBE is used for PAGE of smaller mol. wt. DNA (MW<2000) and
agarose gel electrophoresis of longer DNA.

12. Loading buffer
• This buffer contains glycerol or sucrose to increase the density of
the DNA solutions; otherwise, the samples would dissolve in
running buffer tank and not sink into the gel pocket.
• Loading buffer also contains dyes that facilitate observation of the
sample, e.g. bromophenol blue or xylene cyanol.

13. Voltage/current applied
• The higher the voltage/current, the faster the DNA migrates.
• If the voltage is too high, band streaking may result. Moreover,
high voltage increases the buffer temperature leading to melting of
the gel and decrease bands resolution.
• On the other side, when the voltage is too low, the mobility of small
DNA is reduced and band broadening will occur due to dispersion
and diffusion.
• Therefore, it is highly recommended not exceed 5-8 V/cm and 75
mA for standard size gels.

14. Visualizing the DNA
Ethidium bromide staining (EBS)
• The localization of DNA within the agarose gel
can be determined by staining with fluorescent
ethidium bromide dye under ultraviolet light.
The dye can be included in both, the running buffer tank and the
gel, the gel alone, or the gel can be stained after DNA separation.
• Ethidium bromide is a potent mutagen
Silver staining (SS)
• Silver staining is a highly sensitive method for the visualization of
nucleic acid and protein bands after separation on polyacrylamide
gels. Nucleic acids and proteins binds to silver ions and form a
visible dark brown band on the gel.

15. METHODOLOGY

16. DNA is negatively charged.
+-
Power
DNA
When placed in an electrical field, DNA will migrate toward the positive
pole (anode).
Cathode Anode

17. +-
Power
DNA
How fast will the DNA migrate?
strength of the electrical field, buffer, density of agarose gel…
Size of the DNA!= Small DNA move faster than large DNA
…gel electrophoresis separates DNA according to size
small
large

18. An agarose gel is prepared
by combining agarose
powder and a buffer
solution.
Agarose
Buffer
Flask for boiling

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

20. Gel casting tray & combs

21. 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

22. Agarose Buffer Solution
Combine the agarose powder and buffer solution. Use a flask that is
several times larger than the volume of buffer.

23. Agarose is insoluble at room temperature (left).
The agarose solution is boiled until clear (right).
Gently swirl the solution periodically when heating to allow all the grains of agarose
to dissolve.
***Be careful when boiling - the agarose solution may become superheated and
may boil violently if it has been heated too long.
Melting the Agarose

24. Allow the agarose solution to cool slightly (~60ºC) and then carefully
pour the melted agarose solution into the casting tray. Avoid air
bubbles.
Pouring the gel

25. Each of the gel combs should be submerged in the melted agarose solution.

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

27. Place the gel in the electrophoresis chamber.

28. 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

29. 6X Loading Buffer: 
 Bromophenol Blue (for color)
 Glycerol (for weight)
Sample Preparation
Mix the samples of DNA with the 6X sample loading buffer (w/ tracking
dye). This allows the samples to be seen when loading onto the gel, and
increases the density of the samples, causing them to sink into the gel
wells.

30. Loading the Gel
Carefully place the pipette tip over a well and gently expel the sample.
The sample should sink into the well. Be careful not to puncture the
gel with the pipette tip.

31. 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

32.  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
(-)


33.  100
 200
 300
 1,650
 1,000
 500
 850
 650
 400
 12,000 bp
 5,000
 2,000
DNA Ladder Standard
Inclusion of a DNA ladder (DNAs of know sizes) on the gel
makes it easy to determine the sizes of unknown DNAs.
-
+
DNA
migration
bromophenol blue
Note: bromophenol
blue migrates at
approximately the
same rate as a 300
bp DNA molecule

34. Staining the Gel
• Ethidium bromide binds to DNA and fluoresces under UV light,
allowing the visualization of DNA on a Gel.
• Ethidium bromide can be added to the gel and/or running buffer
before the gel is run or the gel can be stained after it has run.

35. Safer alternatives to Ethidium Bromide
 Methylene Blue
 BioRAD - Bio-Safe DNA Stain
 Ward’s - QUIKView DNA Stain
 Carolina BLU Stain

36. Staining the Gel
• Place the gel in the staining tray containing warm diluted stain.
• Allow the gel to stain for 25-30 minutes.
• To remove excess stain, allow the gel to destain in water.
• Replace water several times for efficient destain.

37. Ethidium Bromide requires an ultraviolet light source to visualize

38. Gel Electrophoresis is used to...
Solve forensic cases
Solve paternity cases
Diagnose genetic diseases
Determine nucleic acids structure.