Gel Electrophoresis of DNADNA凝胶电泳.pptVIP

Gel Electrophoresis of DNA What is Gel Electrophoresis? Electro = flow of electricity, phoresis, from the Greek = to carry across A gel is a colloid, a suspension of tiny particles in a medium, occurring in a solid form, like gelatin Gel electrophoresis refers to the separation of charged particles located in a gel when an electric current is applied Charged particles can include DNA, amino acids, peptides, etc Why do gel electrophoresis? When DNA is cut by restriction enzymes, the result is a mix of pieces of DNA of different lengths It is useful to be able to separate the pieces - I.e. for recovering particular pieces of DNA, for forensic work or for sequencing 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 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 is submerged (submarine gel) in buffer after hardening Also needed are 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 How does it work? DNA is an organic acid, and is negatively charged (remember, DNA for Negative) When the DNA is exposed to an electrical field, the particles migrate toward the positive electrode Smaller pieces of DNA can travel further in a given time than larger pieces 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 gel DNA is prepared by digestion with restriction enzymes Agarose is made to an appropriate thickness (the higher the % agarose, 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’ added