1. DNA Technology: GEL ELECTROHPHORESIS
Ms. Day
Honors Genetics

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. Gel electrophoresis
Separates DNA restriction fragments of different lengths
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. http://www. sumanasinc

5. Restriction Fragment Analysis
Normal  -globin allele
Sickle-cell mutant -globin allele
175 bp
201 bp
Large fragment
DdeI
376 bp
DdeI restriction sites in normal and sickle-cell alleles of
-globin gene.
Electrophoresis of restriction fragments from normal and sickle-cell alleles.
Normal allele
Sickle-cell allele
201 bp 175 bp
(a)
(b)
Is useful for comparing two different DNA molecules, such as two alleles for a gene

6. Agarose Gel Used in Electrophoresis
Widely used in gel electrophoresis technique for the analysis of DNA (or RNA or proteins)
Routinely used (crime scenes, maternity/paternity cases, etc)
Separates molecules based on their rate of movement through a gel under the influence of an electrical current
Agarose gel is NOT agar but feels similar

7. Agarose
Agarose is extracted from seaweed

8. Scanning Electron Micrograph of Agarose Gel (1×1 µm)
Purpose of Agarose Gel Electrophoresis
To separate a mixture of DNA fragments by size using an electrical charge
The gel is a protein matrix (like a sponge with holes; DNA travels through “holes”)
Scanning Electron Micrograph of Agarose Gel (1×1 µm) 
• Agarose is porous,
allowing for the movement of DNA
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

9. How does gel electrophoresis separate DNA fragments?
Gel acts as a strainer to filter DNA by size
DNA fragments are naturally negatively charged due to the phosphate backbone (PO4-3)
DNA fragments of differing sizes will move though the gel at differing rates
larger fragments (more bases) = do not travel as far from wells
smaller fragments (less bases) = travel farther from wells

10. Movement depends on Charge
DNA is negatively charged (because of phosphate backbone)
DNA will be attracted to positively charged poles and repelled from negatively charged ones

11. Movement Depends on Size
Small DNA move faster than larger pieces DNA
Gel electrophoresis separates DNA according to size
Power source supplies the electrical current
DNA + -
Power
small
large
Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.

12. Restriction Enzymes and Plasmid Mapping

13. Restriction Enzyme Digest  different length pieces are made

14. Each well/column is a “DNA fingerprint”
Gel electrophoresis markers (called standards or ladders) are used for size identification of each DNA fragment
Each well/column is a “DNA fingerprint”

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

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

17. combine agarose powder and buffer (ions + H2o) solution into a flask.
#1: Make Gel
combine agarose powder and buffer (ions + H2o) solution into a flask.
Buffer
Flask for boiling 
Agarose

18. Combine the agarose powder FIRST then and buffer solution.

19. Gently swirl the solution to dissolve.
Melting the Agarose B.
Agarose is insoluble at room temperature (left).
The agarose solution is boiled until clear (right).
Gently swirl the solution to dissolve.
Boil the solution
***Be careful when boiling: agarose solution may become superheated and may boil OVER causing a STICKY MESS 

20. Gel casting tray & combs
Tray makes gel
Comb teeth makes wells

21. Preparing the Casting Tray
COMBS CREATE WELLS!!!

22. Pouring the gel D.
Allow the agarose solution to cool slightly (~60ºC) and then carefully pour the melted agarose solution into the casting tray. Avoid air bubble, why?

23. D.
Make sure that the gel combs are submerged in the melted agarose solution but not touching the bottom.

24. E.
When cooled, agarose forms a flexible gel. It appears cloudy in color when completely cooled (~20 minutes). Carefully remove comb (be very, very careful…don’t remove at an angle!).

25. Place the gel in the electrophoresis chamber.

26. Add enough buffer to cover the gel.
DNA
buffer  
wells   
Anode
(positive end)
RED WIRE!
Cathode
(negative end)
BLACK WIRE!
Add enough buffer to cover the gel.
Make sure each well is filled with buffer.
Fxn: Buffer allows electrical current to FLOW through chamber!

27. Loading and Running Gel
After gel is made and place in chamber, DNA will be loaded into well THROUGH buffer.
1st : DNA are cut into fragments using endonucleases
2nd DNA samples are mixed w/ loading dye
FUNCTION:
weigh down DNA into wells
acts as a mobile dye so you can visualize migration
this is why DNA “falls” into wells and you can SEE it move through gel!!!

28. Sample Preparation Samples of DNA need to be mixed with tracking dye.
Allows DNA samples to be seen in the gel
Increases the density of samples, causing them to sink into the gel wells.
Loading Dye:  FUNCTIONS:
 dye to track movement of DNA in gel (color)
 Glycerol (for weight)

29. Loading the Gel
Carefully place the micropipette tip over a well and gently expel the sample. The sample should sink into the well NOT float in the buffer. Be careful not to puncture the gel with the pipette tip.

30. Running the Gel Place cover on electrophoresis chamber
Connect the electrical leads/wires
Be sure the leads are attached correctly - DNA migrates toward the anode (red).
Look for bubbles should form on electrodes when ON

31. Cathode
(-)
End
 wells
DNA
(-) 
Migration
 Tracking/loading dye
Gel
Anode
(+)
End
After the current is applied, make sure the Gel is running in the correct direction. Loading dyewill run in the same direction as the DNA.

32. •Function= allows you to SEE the bands after the gel is made
Staining the Gel
• Ethidium bromide binds to DNA and fluoresces under UV light, allowing the visualization of DNA on a Gel.
YOU ARE USING A QUICK DNA STAIN!!!
•Function= allows you to SEE the bands after the gel is made
***CAUTION! Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times.

33. 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.

34. 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.

35. Methylene blue requires an ultraviolet light source to visualize

36. Review- Know the functions of in Gel Electrophoresis
Restriction enzyme(s)= makes DNA fragments
Agarose gel= separates DNA fragments based on size
Comb= Makes wells to load DNA into
Casting tray= Make agarose gel
Loading dye= Weighs down DNA & tracks DNA movement through gel
Power source= supplies electrical current
Buffer= maintains current
Stain= makes DNA visible in gel

37. 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

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

39. Semilog paper

40. GRAPH THE LADDER/STANDARD…then make a best fit line or curve!
Fragment Length (bp)
Distance migrated (mm)

41. x bp
Fragment Length (bp)
Distance migrated (mm)