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Agarose (Horizontal) Gel Electrophoresis

Agarose (Horizontal) Gel Electrophoresis. Malasian word for seaweed is “agar-agar”. Agarose is derived from red seaweed. Electrophoresis means “carrying with electricity”. 2% agarose. 1% agarose. Agarose Gels.

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Agarose (Horizontal) Gel Electrophoresis

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

  2. 2% agarose 1% agarose Agarose Gels • When agarose is melted and then cooled it contains pores that can act like a sieve. • Increasing agarose concentration decreases pore size. • 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 -- 2,000 bp -- 1,000 bp -- Produce a standard curve 500 bp --

  10. 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.

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