1. Agarose (Horizontal) Gel Electrophoresis Malasian word for seaweed is “agar-agar”. Agarose is derived from red seaweed. Electrophoresis means “carrying with electricity”.

2. Agarose Gels When agarose is melted and then cooled it contains pores that can act like a sieve. Increasing agarose concentration decreases pore size. 1% agarose 2% agarose Increasing agarose concentration limits the size of molecules that can fit through the pores; limits the size range of molecules that can be separated.

3. Agarose Electrophoresis is a DRAG! Negatively charged DNA is pulled through the agarose, from the cathode (-) to the anode (+). The larger the DNA fragment, the slower it travels through the gel due to frictional drag. This relationship is size dependent (not sequence dependent). (-)(+) gel electrophoresis buffer

4. Gel Apparatus The gel is set up for electrophoresis in a tank holding pH buffer. Electrodes apply an electric field:

5. Electrophoresis Buffer Used for casting the agarose gel and filling electrophoresis chamber. Concentration of buffer used in agarose gel and electrophoresis chamber must be same Function of buffer: –Contains salts for conducting electricity from one electrode to the other. –Contains buffer to maintain pH during electrophoretic separation.

6. Loading Buffer/Dye Usually mixed with samples just prior to loading sample in gel. Serves two functions: –Increases density of sample so that it sinks into gel well and stays there until sample is drawn into gel. (glycerol or glucose.) –Contains dye; relatively small and usually negatively charged. Allows us to visualize how far smallest molecules have traveled through agarose gel.

7. Loading Samples and Starting Electrophoresis Load 10 uL of each sample. Use a fresh micropipette tip (yellow) for each sample. Insert red lead into red input (+) of power supply and black lead into black input (-) of power supply. Set power supply to 100 Volts then turn on power. (Should see bubbles forming at end of chamber with black lead (cathode/-). Stop 2/3 dye of gel

8. Visualizing Genetic Differences Between Individuals Position of bands on a DNA agarose gel is based on size (not DNA sequence contained within the fragment.) One band represents millions of DNA fragments ! (-) (+)

9. Standards-Based Size Determination SIZE 10,000 bp 8,000 bp 6,000 bp 4,000 bp 2,000 bp 1,000 bp 500 bp DISTANCE ? mm ? (-) (+) 10,000 bp -- 8,000 bp -- 6,000 bp -- 4,000 bp -- 1,000 bp -- 500 bp -- 2,000 bp -- Produce a standard curve

11. STAINING OF GEL Different stains are used for different classes of macromolecules. DNA and RNA are generally stained with ethidium bromide (EtBr), an intercalating agent. The DNA-EtBr complex fluoresces under UV light. Protein is stained with Coomassie Blue or Silver Stain.