3.8 Gel Electrophoresis

Gel Electrophoresis - technique used to separate DNA fragments based on their size

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

Sample then placed into small plastic tube

DNA in sample will contain many different sized fragments

If we could see them, they would be easily sorted

Too small to be seen and must be detected in another way

Agarose gel used as “filter” to separate fragments based on size

Technique can also be used to separate other organic molecules such as proteins

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

Electric current placed across gel, causing fragments to move away from well

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

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

Stain used to make DNA fragments visible

Commonly used stain - ethidium bromide - inserts itself among complimentary base pairs of DNA and fluoresces under U.V. light

Result is pattern of bands indicating groups of same sized fragments

Length of these fragments can be determined by comparing position of each band to a standard sample of DNA fragments