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Gel 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

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Presentation Transcript
purpose of gel 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
slide3
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
How It Separates
  • The gel is a porous matrix (like a sponge)
  • Separates DNA based on
    • Size
    • Charge
separation by size

start

end

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

Image taken without permission from http://www.dnai.org/b/index.html-- Gel Electrophoresis Animation

separation using charge
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 charge1
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

-

well

Direction DNA travels

-

+

Direction DNA travels

SIDE VIEW

TOP VIEW

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The Gel
  • Wells are created to put the DNA into
  • We use agarose gels to separate DNA
challenges
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
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
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
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
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

(-) start

A

B

(+) end

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)
absolute size
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

1000 bp

850 bp

750 bp

600 bp

200 bp

100 bp

Example

Size Standard

Sample 1

Sample 2

-

  • Suppose you have a size standard with the following sized fragments: 1000 bp, 850 bp, 750 bp, 600 bp, 200 bp, 100 bp
  • Based this info, how big is the circled fragment?
    • 850 bp

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