gel electrophoresis purpose of gel electrophoresis a method for separating dna can be used to...
TRANSCRIPT
Gel ElectrophoresisGel Electrophoresis
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
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
How It Separates
• The gel is a porous matrix (like a sponge)
• Separates DNA based on– Size– Charge
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
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
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
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
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)?
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!
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
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
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
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
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
Example• Suppose you have a
size standard with the following sized fragments: 1000 bp, 850 bp, 750 bp, 600 bp, 200 bp, 100 bp
Size S
tanda
rd
1000 bp850 bp750 bp600 bp
200 bp100 bp
-
+Sa
mpl
e 1Sa
mpl
e 2
• Based this info, how big is the circled fragment?–850 bp