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Molecular Cloning

by Georgina Cornwall, PhD
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    00:00 Now let us talk cloning. What is cloning?.

    00:04 We were not talking about Dolly, the whole sheep clone. We are talking about small pieces of DNA. Cloning was initially the only way we had to amplify or make more of a particular piece of DNA. We may clone in order to create a DNA library, a whole set of clones of all of the DNA in an organism. And we may clone in order to create more copies of a particular piece of DNA, but it is a relatively slow process.

    00:35 Towards the end of the lecture, I will introduce polymerase chain reaction, which speeds the whole thing up. But molecular cloning involves isolation of the gene were interested in, insertion of that gene into a bacterial genome and then amplification by asking the bacteria to amplify themselves as in reproduce and make more copies of the genes we have inserted into their plasmid or their bacterial chromosome. We have a plasmid and this plasmid has two special things in it, maybe three special things. First of all, it needs an origin of replication.

    01:13 Otherwise, we cannot make any copies of it. If it has an origin of replication, then we need to make sure that it has an inserted antibiotic resistance gene. In addition to that, we need a gene that expresses a particular phenotype. Here we have the antibiotic gene in red and this gene that expresses a particular phenotype in yellow. In this case, we will be looking at the lacZ gene, which codes for the enzyme beta-galactosidase. Beta-galactosidase breaks down the lactose sugars. If that gene is functional, then betaglucosidase works, if the gene is not functional, then it does not work. Keep that in the back of your mind.

    01:58 The ampicillin resistance gene is the red piece and ampicillin resistance gene will allow this plasmid or bacteria containing this plasmid to grow when they are on an ampicillin-containing medium. So now, we have the restriction endonuclease that is going to cut specifically in the middle of lacZ gene so that we can tell if the gene of interest that we are inserting has been inserted or not. So that lacZ gene has opened up, we can then insert the foreign DNA.

    02:36 It is going to want to anneal with the bacterial plasmid and then we have a recombined plasmid.

    02:46 Or we don't have a recombined plasmid perhaps the DNA did not make it in there in which case we have a functional betaglucosidase gene and we have an interrupted one down on the bottom with the new DNA of interest in there. Now we need to get this DNA into a bacteria so that the bacteria can reproduce it. We are going to ask the bacteria to pick up the plasmids from the environment. If they do so, then we will take them to grow them on a plate. Some of them keep in mind have picked up the plasmid. Some of them have not picked up the plasmid. Some of them have picked up a plasmid that does not have the DNA of interest in and some of them have picked up of plasmid that does have the DNA of interest in them. So we trick them with this plate of media and the medium is going to contain ampicillin.

    03:42 If the bacteria picked up the plasmid, then it will have ampicillin resistance, the red gene and they will grow on the plate. The other thing that we supplement this plate with its X-gal.

    03:57 X-gal is a sugar that when betaglucosidase is in action, then it will turn blue. We have a phenotypic thing we can observe. We will let our bacteria that have been transformed grow on this plate and if they have an active lacZ gene, the blue color is formed and if they do not have an active gene, then the bacterial cultures grow white. From this result, we can tell that not only has the bacteria taken up the plasmid because otherwise they wouldn't grow on the plate at all, the ampicillin will kill them and also we can tell that it has picked up gene of interest. So which culture we really interested in on this plate? We are interested in the ones that are depicted in yellow that have the broken lacZ gene because we know they have taken up the DNA of interest, whatever that might be. We will look at some examples shortly.


    About the Lecture

    The lecture Molecular Cloning by Georgina Cornwall, PhD is from the course Biotechnology.


    Included Quiz Questions

    1. The vector (plasmid) should have an origin of replication so that multiple copies can be made
    2. An antibiotic resistance gene to allow growth on antibiotic containing medium
    3. A gene with an observable phenotype that can be interrupted by the DNA of interest
    4. An antibiotic resistance gene that can be interrupted by the DNA of interest
    1. The method of replication of one molecule of DNA to produce a population of cells with identical DNA molecules
    2. The method of replication of one molecule of polysaccharide to produce a population of cells with polysaccharide molecules
    3. The method of replication of one molecule of protein to produce a population of cells with protein molecules
    4. The method of replication of one molecule of fatty acid to produce a population of cells with fatty acid molecules
    5. The method of replication of one molecule of phospholipid to produce a population of cells with phospholipid molecules
    1. White bacterial colonies
    2. Blue bacterial colonies
    3. Pale blue bacterial colonies
    4. Metallic blue bacterial colonies
    5. Bacterial colonies with any color except white and blue

    Author of lecture Molecular Cloning

     Georgina Cornwall, PhD

    Georgina Cornwall, PhD


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