Gel Electrophoresis

1. Gel Electrophoresis Resource: http://web.utk.edu/~khughes/GEL/sld001.htm

2. What is Gel Electrophoresis? • Gel Electrophoresis separates molecules on the basis of their charge and size. • The charged macromolecules migrate across a span of gel because they are placed in an electrical field. • The gel acts as a sieve to to retard the passage of molecules according to their size and shape.

3. Organic molecules such as DNA are charged. • DNA is negatively charged because the phosphates (red circles) that form the sugar-phosphate backbone of a DNA molecule have a negative charge.

4. Occur naturally in bacteria to attack viruses Recognize a specific sequence of bases and cut the DNA backbone recognition sites --> palindromes sequences are the same but run in opposite directions first strand: 5’ to 3’ second strand: 3’ to 5’ Enzymes are named from the organism they are isolated from EcoR1 (GAATTC) Sau3A (GATC) Creates fragments of DNA different sizes What are Restriction Enzymes?

5. A gel is prepared which will act as a support for separation of the fragments of DNA. The gel is a jello-like material, usually agarose, a substance derived from seaweed. • Holes, called wells, are created in the gel. These will serve as a reservoir to hold the DNA solution.

6. DNA solutions (mixtures of different sizes of DNA fragments) are loaded in a well in the gel.

7. The gel matrix acts as a sieve for DNA molecules. • Large molecules have difficulty getting through the holes in the matrix. • Small molecules move easily through the holes. • Because of this, large fragments will lag behind small fragments as DNAs migrate through the gel

8. As the separation process continues, the separation between the larger and smaller fragments increases.

9. Molecular weight markers are often electrophoresed with DNAs. • Molecular weight markers are usually a mixture of DNAs with known molecular weights. • Molecular weight markers are used to estimate the sizes of DNA fragments in your DNA sample.

10. DNA Fingerprinting • The resulting pattern is called a DNA fingerprint. • If identical patterns appear in different columns the samples may be from the same source.

11. Loading the Gel • DNA samples are mixed with a "loading dye." The loading dye allows you to see the DNA as you load it and contains glycerol or sucrose to make the DNA sample heavy so that it will sink to the bottom of the well.

12. Running the Gel • Electrical current is applied. • DNA fragments will migrate through the gel at various rates, depending on their size. • When the dye marker indicates that DNA fragments have moved through the gel, the current is turned off and the gel is removed from the tray.

13. Things to think about… • Why does the electrophoresis buffer contains salts? • Why will bubbles (of oxygen) form during electrophoresis? • Hint: Think of the solvent for the electrophoresis buffer…

14. Additional Practice • http://learn.genetics.utah.edu/units/biotech/gel/ • http://www.dnalc.org/ddnalc/resources/electrophoresis.html • http://www.life.uiuc.edu/molbio/geldigest/electro.html • http://www.tvdsb.on.ca/westmin/science/sbioac/genetics/Electro.htm • http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html