Using the techniques of molecular biology to clone genes and analyze DNA fragments using gel electrophoresis.
The following experiment was carried out over a period of three weeks. During the first week a ligation reaction was set up using plasmid DNA that contain the gene for ampicillin and gene from a PCR reaction that has been identified and isolated by the instructor. For the gene to be inserted into the plasmid the plasmid must be cut using the restriction enzyme endonucleases, which cut the internal strand in a strand of DNA. This results in a recombinant DNA molecule. The linearized plasmid vector and gene fragment are then incubated for an hour at temperature with the DNA ligase that causes the vector’s end base pair with each other resulting in regeneration of the circular plasmid without the gene fragment. For the next step of the cloning process bacterial cells grown by the previous lab section will be used to ensure that the cells are capable of taking up DNA. The same bacterial cells are used to grow new cells for the next lab section.
Two bacterial colonies are extracted and dipped in CaCl2. E.coli is a Gram-negative bacterium surrounded by an outer layer and a peptidoglycan layer. For the DNA to penetrate both membranes CaCl2 must be present, allowing the membrane to become porous and allow DNA through the cell wall. The ligation reaction done in the previous step is added to the mixture and incubated on ice for 15 minutes to ensure that the DNA penetrates the cells. The mixture is then put through a process known as heat shock where the temperature is raised to 420C to prevent the cellular protein from denaturing. After another short incubation on ice 1.0 ml LB broth which contained no antibiotics is added to the bacteria. This mixture is then incubated at 370C to give the cells enough time to express the ampicillin resistance gene. After 30 minutes 200 micro... [continues]
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