DNA cloning & Transformation
DNA cloning
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DNA cloning starts when The restriction enzyme EcoR1, cuts the plasmid in one place so that it creates an area that the target DNA can bind to. The target gene (Insulin gene) is cut out of its original DNA strand with the same restriction enzyme EcoR1,so that just the gene of interest is attached to the plasmid for cloning. After cutting both the target DNA and the plasmid, the two are linked together with an enzyme called DNA ligase. This pasting process results in a recombinant DNA plasmid, which is a single DNA molecule combined from two different sources of DNA. It is literally 'recombined,' hence the name 'recombinant.'After the two DNA pieces have been pasted together, the plasmid is inserted into a bacterial cell, which will allow the bacteria to replicate and produce plasmid 'babies' that are identical to the 'parent' plasmid.
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Transformation
transformation is the introduction of foreign DNA, usually by a plasmid into a bacterial cell.
Most bacteria are not naturally competent but can be chemically induced in the laboratory to become so with the aid of calcium chloride. Calcium chloride is a salt that ionizes into Ca21 and Cl2. Bacterial cells are suspended in a solution of calcium chloride at 0°C. The bacterial membrane contains exposed phosphates that are negatively charged. The positively charged calcium ions stabilize the negative charges of the phosphates. In addition, the low temperature “freezes” the cell membrane,which is fluid,making it more rigid. Now that the cell membrane has been stabilized both physically and chemically, the plasmid DNA is introduced into the solution.The negatively charged phosphates of the DNA are also stabilized by the calcium cations.
transformation is the introduction of foreign DNA, usually by a plasmid into a bacterial cell.
Most bacteria are not naturally competent but can be chemically induced in the laboratory to become so with the aid of calcium chloride. Calcium chloride is a salt that ionizes into Ca21 and Cl2. Bacterial cells are suspended in a solution of calcium chloride at 0°C. The bacterial membrane contains exposed phosphates that are negatively charged. The positively charged calcium ions stabilize the negative charges of the phosphates. In addition, the low temperature “freezes” the cell membrane,which is fluid,making it more rigid. Now that the cell membrane has been stabilized both physically and chemically, the plasmid DNA is introduced into the solution.The negatively charged phosphates of the DNA are also stabilized by the calcium cations.
![Picture](/uploads/6/0/8/4/60840857/3528112.png?1457917175)
Now the solution is subjected to a quick heat shock treatment of 42°C that lasts for approximately 90 seconds and creates a draft.The outside environment of the cell is now at a slightly higher temperature than the inside of the cell. The resulting draft sweeps the plasmids into the bacterial cell through pores in its membrane. Finally, the bacterial cells are allowed to recover from the experience. They are incubated in a nutrient media suspension at a temperature of 37 C. Selective plating is a method that can be used to isolate the cells with recombinant DNA. The vectors used for cloning carry an antibiotic-resistance gene. Therefore, if the transformation is a success, the bacteria will be able to grow on media that contain the antibiotic. If no growth is observed, the bacteria were not transformed and were eliminated by the antibiotic.