Bacterial Transformation;DNA Extraction Part I & II:Total Genomic Extraction & Plasmid Extraction;Electrophoresis
Abstract: We conducted three experiments that included a Bacterial Transformation, a two process DNA extraction, and a final procedure using gel electrophoresis. The Bacterial Transformation lab was performed to prepare the plasmid into a bacteria and to use that bacteria to amplify the plasmid in order to make large quantities of it. This is based on the natural function of a plasmid to transfer genetic information vital to the survival of the bacteria. The next process of DNA extraction involved the extraction of total genomic DNA, and extraction of bacterial plasmids. The extraction of DNA is important because it is necessary to separate the DNA from other materials so that we can use the DNA for the final electrophoresis process. The gel electrophoresis portion of the lab is used to separate the DNA by size or charge. DNA Gel electrophoresis is performed for analytical purposes of the bacterial DNA used throughout this experiment.
Introduction: In a series of three lab experiments, we involved three separate linked experiments that started with transforming E.coli bacteria with a special plasmid called the pGLO (+ &-). This is a specialized plasmid that contains an abnormal promoter that is linked to GFP gene from the Aequorea victoria, a jellyfish that contains a transgenic plasmid containing DNA from two.different organisms. We want to speed up the natural process of bacterial transformation weakening the cell membranes of the bacteria through the transformation, transduction, and conjugation of the E.coli. Following this first process of the three step experiment we will being doing the first DNA extraction (Total Genomic Extraction) which involves the goal of separating the DNA from other materials so that they are readily available for further research. We will extract the DNA from our pGLO plasmid (+/-) to be used later on in our experiment for gel electrophoresis. The second DNA extraction process involves the extraction of the bacterial plasmids after purifying the samples from the previous procedure. A similar process related to the previous extraction is going to be performed to remove the plasmid except using a method that is more efficient. This plasmid DNA will also be used in the next process of gel electrophoresis. The final process of DNA gel electrophoresis involves both the total genomic DNA, and the plasmid DNA that was extracted from the +pGLO and -pGLO used in the first process during the transformation experiment. Using two different dyes, we will prep the the samples with these dyes to accomplish two things. The first dye called bromophenol blue, is used to make the loading process when the samples are ready to injected into the gel easier. The second dye called Ethidium Bromide, a hazardous mutagen and carcinogen used to make the samples in the gel more visible for observation and analysis. The purpose of this is to separate the different separate a mixed population of DNA fragments by length, to estimate the size of DNA fragments.
Materials & Methods: In the first process of this experiment of Bacterial Transformation, began with the use of both a negative and positive pGLO. 1 ml of E.coli was added to the +pGLO then pelleted through centrifuging then 500 micro liters of Ca Cl transformation solution was added to the +pGLO tube and re suspended using pipettiing. 250 micro liters of the transformation solution was added to -pGLO tube then both tubes were placed on ice for 30 min. After, 5 micro liters of of suspended plasmid DNA is added to the +pGLO plasmid only. The tubes are placed back on the ice for 10 more mins. A heat shock is used next inside a 42 degree Celsius hot water bath for exactly 50 seconds then moved back to the ice to cool. 250 micro liters of LB nutrient broth is added to each...