第四讲 核酸电泳技术.pptVIP

Gel Electrophoresis a method used to separate macromolecules like proteins and nucleic acids (DNA/RNA) basedon their size and electric charge. Gel Electrophoresis: What is it? How is Gel Electrophoresis Achieved? ? the anode and cathode (+) are at either end of the gel , creating electrical current . these attract or repel molecules depending on their charge. How does it work ? DNA is an organic acid, P skeleton has – charge, negatively charged . When the DNA is exposed to an electrical field, is repelled from the anode (-) and attracted to the cathode (+) . So the particles migrate toward the positive electrode./+ pole The migrating rate of DNA across the gel depends on their size. The smaller segments move/travel faster and farther than larger ones in a given time . DNA appears as a banding pattern What is needed ? Agarose - a polysaccharide made from seaweed. Agarose is dissolved in buffer and heated, then cools to a gelatinous solid with a network of crosslinked molecules. Some gels are made with acrylamide [??kri‘l?maid]丙烯酰胺 if sharper bands are required. Buffer - in this case TBE The buffer provides ions in solution to ensure electrical conductivity. Not only is the agarose dissolved in buffer, but the gel slab半固体 is submerged (submarine gel) in buffer after hardening Also needed is a power supply and a gel chamber Gel chambers come in a variety of models, from commercial through home-made, and a variety of sizes. A gel being run Agarose block Positive electrode DNA loaded in wells in the agarose Black background To make loading wells easier Comb Buffer Steps in running a gelHow is Gel Electrophoresis Done? Agarose is made to an appropriate thickness (the higher the % agarose, the smaller pores are, and the slower the big fragments run) and ‘melted’ in the microwave The gel chamber is set up, the ‘comb’ is inserted The agarose may have a DNA ‘dye’ EB added (or it may be stained later). The agarose is poured onto the gel block and cooled The co