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BIOL 2301

Transformation of E. coli and Analysis of plasmid DNA Melissa Johnson Maggie McCrann Lab Time: Tuesday 2:50 Date Submitted: 11/27/07 Introduction The first exercise in this multi-step experiment was the transformation of E. coli with the plasmid pAMP. The bacteria in the lab were transformed with plasmid DNA containing an ampicillan resistance gene. In genetics, the natural transformation of bacteria is the incorporation of genetic material from a foreign source in the environment (Begley 2007). In this experiment, two steps are needed in order to transform the bacteria. The first step is to prepare competent bacterial cells that can be used for transformation. The next step is the actual transformation of competent cells. This is done by adding the pAMP solution to the competent cells. Six plates were prepared, 3 with ampicillan and 3 without ampicillan. The growth on these plates was observed and recorded. The second exercise was a mini-prep done to prepare plasmid DNA. A mini-prep allows the observer to verify that the transformed phenotype was due to the incorporation of pAMP. The mini-prep isolates the plasmid DNA from the transformed E. coli cells. The basic principles of the mini-prep are to grow, harvest, and lyse transformed cells and then separate the plasmid DNA from the cellular components (Begley 2007). The E. coli suspension was added to two tubes, which were spun to pellet the cells. The glucose solution, potassium acetate, and SDS solution were then added. This lysed the cells, dissolved the organic material and precipitated the proteins from the mixture. The tubes were spun again to pellet the precipitate, and the supernatant was collected. Isopropanol was added and the tubes were spun again to precipitate and pellet the nucleic acids. Ethanol was added to wash the pellets after the supernatant was removed, and then the tubes were spun again. The ethanol was removed and the pellets were resuspended. In the third exercise of this experiment, a restriction analysis of the mini-prep DNA was carried out. The purpose of this laboratory is to compare fragments of the plasmid DNA with sample pAMP fragments to verify that the plasmid contains the same restriction sites (Begley 2007). The plasmid is cut with two restriction enzymes. Because we know the location of the restriction sites for these enzymes on pAMP, and we can predict the size of the fragments expected from the plasmid, we can verify that the plasmid contains the pAMP gene. Four tubes are prepared for this experiment. Gel electrophoresis is carried out on these four samples and the results are observed and recorded below. Gel electrophoresis is a useful tool in the genetics laboratory because it allows you to separate miniscule components of a solution based on their size (Begley 2007). The further they go in the gel, the smaller the molecules are. Results Exercise 1: Transformation of bacteria Plate Medium Luria Broth X (+pAMP) Y(-pAMP) Growth A LB-Amp 90µL 10µL - 1 colony B LB-Amp - 100µL - 12 colonies C LB-Amp - - 100µL No growth D LB 90µL 10µL - Lawn E LB - 100µL - Lawn F LB - - 100µL Lawn The plate containing ampicillan and 10 micro liters of ampicillan-resistant bacteria had only 1 colony; however the plate containing 100 micro liters of pAMP bacteria had 12 colonies. The plate containing the ampicillan and non-resistant had no growth, as expected. The plates containing no ampicillan and both types of bacteria each had a lawn of gro
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