1. DNA Technology: GEL ELECTROHPHORESIS
Ms. Day
AP Biology

2. DNA Gel Electrophoresis
DNA fingerprint
**Each band that you see is a collection of millions of
DNA molecules, all of the same length!!
Restriction Fragment Analysis
detects DNA differences that affect restriction sites

3. What is Gel Electrophoresis?
Process that separates DNA restriction fragments of different lengths
DNA cut with restriction enzymes
Uses electrical current to separate DNA based on size
DNA has a negative charge.
DNA moves towards the POSITIVE electrode. Why?
DNA molecules of SMALLER sizes move the furthest through the gel.

4. Movement depends on Charge
DNA is negatively charged (because of phosphate backbone)

5. Purpose of Agarose Gel Electrophoresis:
Gel electrophoresis separates a mixture of DNA fragments according to size
Uses an agarose gel to do this
Made of sugar from seaweed
Agarose gel acts as a strainer to filter DNA fragments based on size
a gel contains a protein matrix
It is porous
Has “holes” like a sponge
Now it would be great to make a picture of many lines of various sizes mixed up on one size and organize according to size in the other
Scanning Electron Micrograph of Agarose Gel (1×1 µm) 

6. Agarose: Agarose is a linear polymer extracted from seaweed.
D-galactose
3,6-anhydro
L-galactose
Sweetened agarose gels have been eaten in the Far East since the 17th century.
Agarose was first used in biology when Robert Koch* used it as a culture medium for Tuberculosis bacteria in 1882
Can be used to separate DNA fragments > 300 bp
Agarose is a linear polymer extracted from seaweed.

7. http://www. sumanasinc

8. How does gel electrophoresis separate DNA fragments?
DNA fragments of differing sizes will move though the gel at differing rates (speeds)
larger fragments = more bases  do not travel as far as smaller fragments containing less bases
Larger fragments get “stuck” in gel sooner than smaller pieces

9. Movement Depends on Size:
Small DNA move faster than large DNA
…gel electrophoresis separates DNA according to size
DNA + -
Power
small
large
Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.

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

11. Electrophoresis Apparatus
Making an Agarose Gel
And Setting up your Gel
Electrophoresis Apparatus

12. ions in aqueous solution.
Combine:
agarose powder
a buffer
ions in aqueous solution.
Buffer
Flask for boiling
Agarose

13. Use a flask that is several times larger than the volume of buffer.
Agarose
Buffer Solution
Use a flask that is several times larger than the volume of buffer.

14. Agarose is insoluble (cloudy) at room temp
Melting the Agarose
Agarose is insoluble (cloudy) at room temp
Agarose solution is boiled until clear (right).
Gently swirl the solution
***Be careful when boiling
agarose solution may become superheated and boil violently over  A SUGARY MESS 

15. Gel casting tray & combs

16. Preparing the Casting Tray
Seal edges of casting tray with black plastic sides
Put in 1 comb with teeth  will create wells for DNA samples
Place the casting tray on a level surface
Pour molted gel into casting tray BE CAREFUL  HOT!!!

17. Avoid air bubbles when pouring (“casting”) gels, why?
Pouring the gel
Avoid air bubbles when pouring (“casting”) gels, why?

18. Make sure gel comb teeth are submerged in melted agarose solution…why?

19. When cooled, the agarose polymerizes, forming a flexible gel
When cooled, the agarose polymerizes, forming a flexible gel. It should appear lighter in color when completely cooled (30-45 minutes). Carefully remove the comb (be very, very careful!).

20. Making Wells…
After agarose solidifies, comb is removed leaving wells where the DNA will be loaded.
Remove comb by pulling up at 90° angle
Comb teeth create wells, where DNA is placed

21. Place the gel in the electrophoresis chamber.

22. Add buffer to cover the gel to a depth of at least 1 mm.
DNA
buffer  
wells   
Anode
(positive end)
RED WIRE!
Cathode
(negative end)
BLACK WIRE!
Add buffer to cover the gel to a depth of at least 1 mm.
Make sure each well is filled with buffer.

23. DNA Sample Preparation
1. DNA samples are digested using restriction enzymes  Fragments are made!
2. Mix the samples of DNA with loading solution (w/ tracking dye). This allows:
DNA samples to be seen when loading and running gel
Increases density of DNA samples, causing them to sink into the gel wells.
Mixture contains…
 Bromophenol Blue (for color)
 Glycerol (for weight)

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

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

26. Cathode
(-)
End
DNA
(-) 
Migration
 wells
 Bromophenol Blue
Gel
Anode
(+)
End
After current is applied, make sure the gel is running in the correct direction.
Tracking dye will run in the same direction as the DNA just ahead of it

27. **STAIN is different than LOADING DYE
Staining the Gel
• Stain binds to DNA and fluoresces under UV light, allowing visualization of DNA bands on gel.
BUT…..YOU ARE USING A QUICK DNA STAIN!!!
**STAIN is different than LOADING DYE
***CAUTION! Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times.

28. • Place the gel in the staining tray
Staining the Gel
• Place the gel in the staining tray
Slide it in from casting tray AFTER gel is run

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

30. Methylene blue requires an ultraviolet light source to visualize

31. Visualizing the DNA
 100
 200
 300
 1,650
 1,000
 500
 850
 650
 400
5,000 bp
 2,000
DNA ladder 
DNA:
wells
Samples # 1, 4, 6 & 7 were positive for DNA samples taken
from the crime and compared to suspect

32. Samples # 1, 6, 7, 10 & 12 were positive for our suspect and
Visualizing the DNA (Actual Image)
DNA ladder 
wells
DNA
 2,000 bp
 1,500
 1,000
 750
 500
 250
Samples # 1, 6, 7, 10 & 12 were positive for our suspect and
crime scene samples
March 12, 2006

33. Movement of DNA fragments in agarose gels
There is a linear relationship between”
the migration rate of a DNA fragment and
The size of the fragment (in basepairs).
Larger molecules move more slowly through the gel because of more friction

34. Semilog paper

35. You have to graph the STANDARD
marker DNA fingerprint on the semilog paper
Fragment Length (bp)
Distance migrated (mm)

36. Then you USE the graph to find the UNKNOWN fragment lengths!
x bp this is the length of the unknown fragment
Fragment Length (bp)
Distance migrated (mm)