Transformation Of Escherichia Coli With pGLO Plasmid
April 24, 2013
ABSTRACT: This experiment focuses on genetic engineering and transformation of bacteria. The characteristics of bacteria are altered from an external source to allow them to express a new trait, in this case antibiotic resistance. In is experiment foreign DNA is inserted into Escherichia coli in order to alter its phenotype. The goal of the experiment is to transform E. coli with pGLO plasmid, which carries a gene for ampicillin resistance, and determine the transformation efficiency. The bacteria are transformed by a combination of calcium chloride and heat shock. When the bacteria are incubated on ice, the fluid cell membrane is slowed and then the heat shock
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The tubes were then placed back on ice. Ten nanograms of pGLO DNA solution was then added to one of the tubes and the tubes were incubated on ice for ten minutes. While the tubes were incubating, four plates of LB nutrient agar were obtained, one containing just LB, two also with ampicillin, and one also with ampicillin and arabinose. The test tubes after the ten minutes were incubated for 50 seconds in a 42 degree Celsius water bath and then returned to the ice for another two minutes. The tubes were then removed from the ice and 250 microliters of LB nutrient broth was added to each tube, which was then incubated for an additional ten minutes at room temperature. 100 microliters of the solution in the tube containing pGLO was added to one plate with just LB agar and ampicillin and to the plate with ampicillin and arabinose. 100 microliters of the solution in the tube without pGLO was added to the other plate containing LB and ampicillin as well as to the plate containing only LB. The solution was spread around the surface of each agar using a new sterile loop for each plate. The plates were then incubated at 37 degrees Celsius and results were observed and recorded after 27 hours.
Yellow colonies; still yellow under UV light
Yellow colonies; glow green under UV light
Table 1. Transformed Plates After Incubation shows that there was growth of transformed bacteria