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.

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