Saturday, April 6, 2019

Gel electrophoresis Essay Example for Free

Gel electrophoresis EssayThe explosion of molecular biology techniques that began in the mid-1970s (and continues today) has provided tools to taste the physical structure of deoxyribonucleic acid, its nucleotide sequence and how genes are read and regulated. One key tool is the dexterity to visualize deoxyribonucleic acid grains and determine their length by using a technique called jelly electrophoresis. submission to gelatin electrophoresisIn gel electrophoresis, DNA fragments move through a porous hyaloplasm made of agarose, a gelatin-like substance purified from seaweed. The agarose is melted like Jell-O and then poured into a plastic tray to season into a slab called a gel. A plastic comb inserted at one end mend the gel is hardening forms wells where DNA hears can be pointd. The DNA is miscellanyed with a loading devotee that contains glycerolthis fabricates it heavier than water, so it will sink to the bottom of the well. The gel is then covered with a w eaken solution that can carry electric current, and electrodes are placed at each end of the gel and connected to a power supply. Because DNA is negatively charged (each nucleotide has a negatively charged phosphate attached to it), it will move toward the positive electrode.Larger molecules move through the agarose more slowly, while smaller onescan slip through the pores faster. So, the fragments wind up arranged in order accord to size, with the smaller ones having moved farther toward the positive pole. Figure 47 shows an example. Because the DNA is invisible, the loading buf fer also contains ii dy e s bromophenol blue (a small dye molecule that behaves like a DNA fragment almost 600 bases long) and xylene cyanol (a larger dye that acts like a DNA fragment of about 4000 bases). These dyes form lines that give you an idea of how far your DNA has moved. Some loading buffers also have a third dye, behaving like a very small DNA molecule (50 bases or so).As the DNA migrates, t he different fragments will form bands each band is composed of many identical copies of a particular-size bandage of DNA (you cant do gel electrophoresis with one DNA molecule you require millions or billions of identical molecules). The last step is to make the DNA bands visible, using a fluorescent molecule that inserts between the bases in the DNA helix. We use a commercial loading buffer called EZ-Vision which includes the fluorescent molecule, so the gel is already stained when its done running. Another method is to soak the gel in ethidium bromide after running it. Either way, the bands can be seen using ultraviolet light and photographed to make a permanent record.Sample preparationOf course, gel electrophoresis requires some kind of DNA examplea plasmid, a PCR product, a segment of a chromosome, etc. If the molecule is circular, enzymes are used to crop the DNA (see the section on restriction digestion, page 87), because circular molecules can be either tightly or loose ly coiled and dont wind up at the same place on a gel as a linear molecule of the same size. Whatever your sample is, it must be mixed with loading buffer (containing glycerol and dyes, as described above) before electrophoresis. tote up a volume of loading buffer equal to 1/5 the volume of your sample and mix it well before loa

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