Gel Electrophoresis

by Georgina Cornwall, PhD

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    00:01 Then we need to think about what these restriction enzymes are used for. We can create recombinant DNA, but we can also cut pieces of DNA into all sorts of different sizes. For example, we might want to do that with DNA fingerprinting. We will look at lot closely at that technique coming shortly. But first let us just look at another technique, which is going to be gel electrophoresis. Let us say we have cut up a bacterial genome or a plant genome into a number of different size fragments and we want to be able to see these fragments, how on earth can we do that? So let's say we have a reaction 1 where we have one restriction enzyme that makes this particular cut site. We have a short fragment and a longer fragment.

    00:53 Then in another restriction enzyme in another mixture, we have a cut side in the second spot, a different restriction enzyme makes another couple of different size fragments.

    01:03 Imagine that this is a whole genome. We have lots and lots and lots of fragments that are cut by these enzymes, then we could have the third one. For example, that cuts in a different place. We have lots of different size fragments from these restriction endonucleases running around cutting it up. Type II had the sticky ends in the palindromic sequence thing, but other restriction enzymes don't have necessarily as much specificity, but they make a cut in specific regions as we will learn later on.

    01:35 What happens? So that we can see this gel electrophoresis is the technique that we will see over and over again utilized in multiple DNA technologies. The basic principle here is we have the three mixtures that we have already looked at and different lengths of fragments of DNA there, but we can't see DNA with the microscopes, so we need to figure out a way to visualize it. Gel electrophoresis just does this. We put the gel in a bath of buffer and there is a current running across the gel. So we have a positively charged anode and a negatively charged cathode. Now if you recall from looking at DNA structure, DNA is fairly negatively charged. When you put it in a current, the negatively charged DNA wants to run towards the positively charged anode. The small fragments can move through the gel much more rapidly than the larger fragments much like if you are screening rocks. You are down by the river and the small rocks will fall through the screen and the larger rocks will get caught behind. The DNA molecules will move through that medium, some moving faster than others because they have to make their way through this gel matrix. If you were to leave this on too long, keep in mind you can't see the DNA fragments. Potentially all of DNA your fragments could run right off the end in which case you go through all of this effort and visualization, which wie'll learn about coming up and you don't see any DNA at all. Quite frustrating. It is very important that a specific amount of time is observed that you know that the fragments are not going through run off the edge, but enough time so that they do separate from each other. In this mechanism, DNA is separated by size and again we will use DNA electrophoresis or gel electrophoresis in order to visualize DNA fragments, protein fragments, RNA fragments.

    03:49 This is a very familiar technique to molecular biologists. Now keeping in mind, the DNA is invisible. We have it in our gel and we have wells at the top of the gel. Our DNA fragments have separated by size, shorter fragments went further, longer fragments did not go as far, but we can't see them at all. A number of different techniques are used to visualize them. The first and most simple is by simply exposing the DNA to a fluorescent dye that attaches to DNA. We can look at that fluorescence and see where the bands of DNA are relative to each other. Again gel electrophoresis is a technique that we will see frequently in order to observe DNA patterns, restriction, fragment length, polymorphism, short tandem repeats, DNA fingerprinting and such.

    About the Lecture

    The lecture Gel Electrophoresis by Georgina Cornwall, PhD is from the course Biotechnology.

    Included Quiz Questions

    1. Add electric current
    2. Turn off the electric current soon enough
    3. Include a DNA dye
    4. Include an RNA probe
    1. The charged molecules like DNA, RNA, and proteins according to their sizes under the influence of electric current
    2. The uncharged molecules like DNA and RNA according to the number of oxygen atoms present in their molecular structure
    3. The charged molecules like DNA and RNA according to the number of hydrogen atoms present in their molecular structure
    4. The uncharged molecules like DNA and RNA according to the number of phosphate atoms present in their molecular structure
    5. The uncharged molecules like DNA and RNA according to the number of nitrogen atoms present in their molecular structure

    Author of lecture Gel Electrophoresis

     Georgina Cornwall, PhD

    Georgina Cornwall, PhD

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