3.8 Gel Electrophoresis

What is Gel Electrophoresis?

technique used to separate DNA fragments based on their size

What gel is used in Gel Electrophoresis?

Agarose gel

What is the first step of Gel Electrophoresis?

Sample of DNA extracted from tissue and exposed to one or more restriction enzymes - cut DNA at specific recognition sites

What is the second step of Gel Electrophoresis? (after restriction enzymes)

Sample then placed into small plastic tube

What does Gel Electrophoresis allow us to do? (in terms of size)

• DNA in sample will contain many different sized fragments

• However, they are too small to be seen and must be detected in another way

• If we could see them, they would be easily sorted

What does Agarose Gel actually help us do?`

used as “filter” to separate fragments based on size

Can Gel Electrophoresis be used on other molecules? If so, give an example.

• YES!

• proteins

What is the third step of Gel Electrophoresis?

DNA fragments place in “wells” at one end of agarose gel

What causes fragments to move across the gel? Why is DNA able to move in this way?

• Electric current paced across the gel

• because phosphate groups in DNA carry a negative charge, which is attracted to positive electrode

Describe the relationship between small, same size, and large fragments. Are groups of fragments visible by this point?

• Smaller fragments move through gel more easily than larger fragments - travel farther along gel

• Fragments of same size move at same speed and group together

• Groups of fragments are still not visible

What makes DNA visible? What is the most common used stain? Why? What is the result?

1. stains

2. ethidium bromide

3. because inserts itself among complimentary base pairs of DNA and fluoresces under U.V. light

4. Result is pattern of bands indicating groups of same sized fragments

How do you determine the lengths of the fragment?

by comparing position of each band to a standard sample of DNA fragments