![]() They recognize specific sequences and then we can figure out well which restrictionĮnzyme should we use to cut out different pieces of DNA, but we have gotten to that Identify these enzymes and we know at what points Have gotten to the point that we can find and Restriction enzymes, and I personally find it fascinating that we as a civilization And the way we do that is using restriction enzymes. Well, the first thing we wanna do is we wanna cut this gene out some how. This is the double-stranded DNA and let's say that this part of this DNA has a gene that we want to clone. Actually, let me just draw, let me just try to draw the two strands just so we remind ourselves. I don't want to have to take the trouble of keep drawing the multiple strands. Strand of DNA right over here and I'm just drawing it as a long, but this is a double-stranded, and I'll just write it down, this is double stranded. So how do we do that? Well let's say that this is a But when we talk aboutĬloning and DNA cloning we're talking about somethingĪ little bit simpler. Or an organism, like a sheep, well then you are creating an animal that has the exact genetic Heard the term cloning in terms of the Clone Wars in Star Wars or Dolly the sheep and that is a related idea. And usually it's a piece of DNA that codes for something we care about, it is a gene that willĮxpress itself as a protein that we think is useful in some way. Which is all about making identical copies of a piece of DNA. Lipopolysaccharides are only present in Gram-negative bacteria like E.coli Lipopolysaccharides are molecules on the outer leaflet of the second plasma membrane. So its structure is : plasma membrane, thinner call wall than Gram-Positive, another plasma membrane, capsule. Gram-negative bacteria have 2 plasma membranes (unlike Gram-positive that have 1). Why lipopolysaccharides are in the way for Gram negative bacteria: The plasmid DNA can then pass into the cell upon heat shock"Į.coli is a Gram-negative bacteria so has lipopolysaccharides, therefore it is required to use the calcium chloride to allow the plasmid with DNA to pass through and be accepted. Positively charged calcium ions attract both the negatively charged DNA backbone and the negatively charged groups in the lipopolysaccharides inner core. "The addition of calcium chloride to a cell suspension (state in which the particles of a substance are dispersed but not totally dissolved in a fluid) promotes the binding of plasmid DNA to lipopolysaccharides. coli competent (able to take up extracellular DNA from the environment in order to perform transformation ). The smaller DNA fragments will move further down because of the complex sugar structure of Agarose gel letting smaller DNA pieces to pass more easily. Then, the separated bands of DNA are cut out from the Agarose gel and then extracted from the gel piece using elution (process of extracting one material from another by washing with a solvent)Ĭalcium chloride makes E. The medium is Agarose gel, so DNA fragments separate according to their size though the sieving effect produced by the Agarose gel. ![]() Since DNA fragments are negatively charged molecules (because the phosphate group has a negative charge), they can be separated by forcing them to move towards the anode (positively charged electrode, attracting the negative charge) under an electric field through a medium. The process of separating and isolating DNA fragments after the sequence was cut off:ĭNA fragments can be separated by gel electrophoresis. “Recombinant vector molecule cannot be created unless the vector and source DNA is cut with the same restriction enzyme” That way, it is easy to pair up the different DNA in the vector molecule with the DNA sequence because they have the same sticky ends If cut by the same type of restriction enzyme, they will have the same type of sticky ends (because the restriction enzyme recognizes only a certain type of recognition sequence). ![]() These enzymes predictably cut both strands because the sequences they recognize are palindromic”ĭNA ligase enzymes can be used to stick the cut DNA pieces back to each other or to a new DNA sequence ![]() “Restriction enzymes cut double-stranded DNA at specific locations based the pattern of bases found at those locations. Restriction enzymes recognize where to cut by recognizing certain 6 base pair recognition sequences (for example, GAATTC). ![]()
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