Lab 3 Transformation and Restriction Mapping (1)

Lab 3 Transformation and Restriction Mapping (1) -...

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Experiment 3: Transformation and Restriction Mapping Zachary Bergman Partner: Anuj Shah 3/3/09 Biology 210-2
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Introduction The goal of this lab was take advantage of bacterial transformation and insert foreign DNA, in this case in the form of a plasmid, into E. coli host cells. After the integration of the plasmid, the host cells are plated on three plates, one plate containing the anti-biotic ampicillin, one plate containing the anti-biotic kanamycin, and a control plate containing no anti-biotic. After a screening process which determines which drug resistant marker the plasmid contains, a single colony from whichever anti- biotic containing plate developed growth is inoculated and allowed to multiply. The new hosts cells, which are now integrated with the plasmid DNA, are then analyzed using restriction digest enzymes to cut the plasmid followed by gel electrophoresis. Analysis of the gel electrophoresis allows us to create a restriction map of the circular plasmid based on the sites at which the DNA was cut. This process is designed to teach the process of plasmid transformation, isolation, and restriction mapping. Materials and Methods Part I: Transformation: This is the step at which the DNA plasmid is inserted into the E. coli host cells and they are screened for their drug resistant markers. After obtaining the E. coli host cells the DNA plasmid was inserted by doing a series of centrifuge steps interspersed with adding DMSO, freezing then thawing, and incubating the cells to damage the cell and allow the plasmid to enter. After this process the cells are then plated on three petri dishes, one plate containing the anti-biotic ampicillin (amp), one plate containing the anti-biotic kanamycin (kan), and a control plate containing no anti- biotic. The plates were then incubated for 1-2 days. Part IIA: Overnight Culture: This step was done 24 hours before the rest of Part II because there need to be time for the host cells to multiply. First, the two petri dishes containing anti-biotics were checked for growth, and a single colony from whichever plate contained colonies was inoculated in a premade broth which allowed the newly formed host cells to quickly multiply and provide enough DNA plasmid for the gel electrophoresis described in a later step. For plasmid B, this broth was a broth that supported the ampicillin resistant marker. Part IIB: Isolation:
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This note was uploaded on 02/16/2012 for the course BIOL_SCI 210-2 taught by Professor Somebody during the Winter '09 term at Northwestern.

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Lab 3 Transformation and Restriction Mapping (1) -...

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