1. Gel Electrophoresis

2. Purpose of Gel Electrophoresis
A method for separating DNA
Can be used to separate the size of
DNA
RNA
Protein
We will be using it to separate DNA

3. DNA
What you start with: A variety of different fragments of DNA all mixed together
We will use gel electrophoresis to separate/sort these fragments

4. How It Separates The gel is a porous matrix (like a sponge)
Separates DNA based on
Size
Charge

5. Separation by Size
As DNA is moved through the gel, smaller sized fragments move through faster than larger sized fragments
Ex. A 100 base pair fragment will move through the gel faster than a 500 bp fragment
start
end
Image taken without permission from Gel Electrophoresis Animation

6. Separation Using Charge
The charge on DNA is what makes it move through the gel
DNA is a charged molecule. What is the charge on DNA?
Negative charge
Why?
Phosphate group is negatively charged
Image taken without permission from Gel Electrophoresis Animation

7. Separation Using Charge
The gel is hooked up to a power source
DNA is loaded into the gel on the cathode (-) end
Gel is placed in a buffer solution that will conduct electricity
Electric current is run through the gel
DNA is attracted to the + end (anode) = “runs to the red”
Image taken without permission from Gel Electrophoresis Animation

8. The Gel Wells are created to put the DNA into
We use agarose gels to separate DNA
SIDE VIEW + -
well
TOP VIEW
wells
Direction DNA travels

9. Challenges
DNA is colorless-- how will we see it on the gel & when we are loading it into the gel?
How do we get the DNA to stay in the well (not float away)?

10. Solution #1
Problem #1: How can we see the DNA sample as we load it into the gel
Problem #2: How can we make sure DNA won’t float away
Solution: Add loading dye to the initial DNA sample!

11. Loading Dye
Adds mass to the DNA sample so that it will go into the well
makes it sink to the bottom
Adds blue color so you can see what you are pipetting

12. Solution #2
Problem: DNA is colorless. Once the DNA has been run through the gel, how can we see where it is on the gel?
Solution: Add Ethidium Bromide (EtBr) or Gel Red to the gel

13. Ethidium Bromide The DNA intercalates with the Ethidium Bromide (EtBr)
Intercalates = inserts itself between bases
GelRed also stains nucleic acids
EtBr and GelRed will fluoresce under UV light
GelRed is safe because it has been engineered so that it cannot cross the cell membrane.

14. Relative Size vs. Absolute Size
(+) end
(-) start
Looking at a gel, you can determine which fragments of DNA are bigger than others = Relative Size
Which fragment is bigger, A or B?
Fragment A (didn’t travel as far in a fixed amount of time)

15. Absolute Size
How can we determine the actual size of the DNA fragments (how many base pairs- bp)?
Use a size standard
Also called a DNA ladder
Consists of a series of fragments of known sizes
Use it to compare to your DNA fragments

16. Example
Size Standard
Sample 1
Sample 2
Suppose you have a size standard with the following sized fragments: bp, 850 bp, 750 bp, 600 bp, 200 bp, 100 bp -
1000 bp
850 bp
750 bp
600 bp
Based this info, how big is the circled fragment?
850 bp
200 bp
100 bp +