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Plasmid Fusion And Pcr Essay

1159 words - 5 pages

This details the molecular biology lab in AP Biology. Best grade in class.The AMGEN Lab that we have been doing for the past two weeks consisted oftwo parts; Plasmid Fusion and PCR. Each one is a complicated procedure of geneticengineering, with our own cheek cells and E.Coli supplied by AMGEN. I will start byexplaining the Plasmid Fusion lab.The Plasmid Fusion lab consisted of four major parts; plasmid digestion, gelelectrophoresis, restriction enzyme inactivation and ligation, and the final step, plating out.But, before I get into that I should define some parts of the lab. The main pieces of geneticinformation we will be working with are plasmids. Plasmids are gene sequences found in aloop outside of the main chromosome. Their main purpose is to code enzymes that digestantibiotic enzymes. Antibiotics are chemicals that kill bacteria or interfere with theirgrowth or metabolism. Cells that have antibiotic resistance have an advantage becausethey are able to grow in places that other cells can not.Our main purpose in this lab is to give an E.Coli cell immunity to the antibacterials,Ampicillin and Chloramphenicol by genetically doctoring its plasmids. The first step indoing this is to "cut" the plasmids so that we can ligate the new pieces on later. The DNAwill be cut once twice or not at all because the process does not work all of the time withall DNA. The part that makes the resistance enzyme will be left in tact and separated into asmaller section of DNA. We do this so that we can isolate the genome so that we can laterattach other sections of DNA to it. The next step involves checking to see if theRestriction Enzymes did their job by a process called Electrophoresis. First we suspendthe DNA in a solution of Agarose. Then an electric is applied, one end is "+" and the other"-". DNA forms ions, like most substances that dissolve in water. It separates into H+ andDNA-. The DNA will move toward the + end through the Agarose. Since the Agarosewill stop many of the bigger pieces but the pieces that were cut will pass through becausethey are smaller. Using a chemical called Ethidium Bromide (EtBr) and UV light we cansee how far our DNA made the journey. The farther it went the smaller the pieces and thesmaller the pieces the better the Restriction Enzymes worked. The next step involves threechemicals; the two separated plasmids and a T4 ligator. Mixing these three togethershould form one final strand. The two pieces will be ligated to form the correct genome.The next step is to put the new resistant strand of DNA back into the E.Coli. This processinvolves mainly hot and cold water. The DNA will be put in a iced solution with the cells.The cells should have small holes in them to let the plasmid back in. They will then beshocked by putting them in a hot water bath for two minutes and then put back into theice. The shock should open up the cell long enough for the plasmids to get through. If theplasmid makes it through, the cell should accept the new...

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