1. Gel Electrophoresis

2. What is Gel Electrophoresis?
Electro- electricity
-phoresis-to carry across
A technique used to separate and analyze DNA and proteins
Can be separated based on charge (positive vs. negative) or size
The standard method for DNA is to use a gel that is made of agarose
Agarose is a protein that is obtained from seaweed
It is dissolved in boiling solution and then as it cools back down, it forms a gel

3. Why do we do it?
After DNA is cut with restriction enzymes, there is a mix of DNA fragments of all different sizes
Review: What is a restriction enzyme?
Using gel electrophoresis, the DNA can then be separated by size and further analyzed
This is used for:
Sequencing and analyzing a DNA strand
DNA fingerprinting
Diagnosis of genetic disorders

4. How does it work? DNA is a negatively charged molecule
The gel is exposed to an electrical current and the negatively charged DNA molecules will move towards the positive electrical charge
The gel is like a sponge in the sense that there are pores (holes) in the gel for the DNA to travel through

5. The speed that the DNA moves is dependent on the size of the piece of DNA
The smaller DNA fragments move faster: they have an easier time navigating through the holes and moving down the gel and will finish closer to the positive end
The larger DNA fragments move slower, ends closer to the negative

6. How is it set up?
Once the agarose has been dissolved, it is poured into a mold to settle
While still in the liquid form, a comb is placed into the gel
Once the gel is hardened and the comb is removed, there are wells, or holes, that the DNA can be put into
The gel is placed into a solution (buffer) that will help conduct the electricity

7. Gel Mold and Combs

8. Electrophoresis Setup
DNA
buffer  
wells
Positive 
End
 Negative
End

9. Preparing the DNA
DNA is mixed with dye (so it can be seen while loading) and glycerol (to make it sink)

10. Loading the DNA into the Gel
DNA is added to the wells
Carefully added to the well with a pipette so that a hole is not poked in the gel
The DNA should sink to the bottom

11. Electricity is applied to gel and the DNA begins to move

12. ~~~~~~~~~~~~~~~~~~~~~~~~
Agarose gel
Negative
Positive
DNA fragments
buffer
~~~~~~~~~~~~~~~~~~~~~~~~
Negative
Positive
current
buffer
~~~~~~~~~~~~~~~~~~~~~~~~

13. Gel Electrophoresis Video

14. How is the DNA measured?
 100
 200
 300
 1,650
 1,000
 500
 850
 650
 400
 12,000 bp
 5,000
 2,000
DNA
migration
Usually at least one well of the gel will be loaded with a known DNA sequence called a marker
Acts as a ruler for DNA fragments
Unknown DNA fragments can then be compared to this marker band, or DNA ladder, to figure out the size

15. How is the DNA seen?
After the gel is finished running, it must be stained so that DNA can be seen by the naked eye
Individual DNA strands cannot be seen, but groups of DNA fragments of the same size can
Brighter/darker bands
indicate more DNA
* The original dye separates from the DNA strands during the electrophoresis process