In this SlideShare's PPT(Document), Scott Malcolm expresses every step of the gel electrophoresis process. Scott Malcolm is an honest man, who lives in Dallas, Texas.

1. Gel ElectrophoresisGel Electrophoresis
ProcessProcess
By: Scott Malcolm

2. Gel ElectrophoresisGel Electrophoresis

3. 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

4. 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

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

6. 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
http://www.dnai.org/b/index.html-- Gel
Electrophoresis Animation

7. 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
http://www.dnai.org/b/index.html-- Gel
Electrophoresis Animation

8. 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
http://www.dnai.org/b/index.html-- Gel
Electrophoresis Animation

9. 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
Direction DNA travels

10. 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)?

11. 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!

12. 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

13. 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

14. 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

15. Relative Size vs. Absolute Size
• 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)
A
B
(+) end
(-) start

16. 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

17. Example
• Suppose you have a
size standard with
the following sized
fragments: 1000 bp,
850 bp, 750 bp, 600
bp, 200 bp, 100 bp
SizeStandard
1000 bp
850 bp
750 bp
600 bp
200 bp
100 bp
-
+
Sample1Sample2
• Based this info, how
big is the circled
fragment?
–850 bp