Gel electrophoresis
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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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Gel Electrophoresis

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Gel electrophoresis

Gel Electrophoresis


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


Gel electrophoresis

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

+

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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