Laboratory Procedure for pGREEN Steps 1. Mark the sterile 15mL tubes with the correct labelings. (LB+plasmid‚ LB/Amp+plasmid). 2. Use a sterile transfer pipet to add 250mL of ice cold calcium chloride to each tube. 3. Place both tubes on ice. 4. Use a sterile plastic inoculating tube to transfer isolated colonies of E.coli from the starter plate to the +plasmid tube. 5. Immediately suspend the cells by repeatedly pipetting in and out with a sterile transfer pipet
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foreign plasmid in order to make large quantities of it. In transforming bacteria‚ a foreign DNA is first joined to a small circular DNA molecule known as a plasmid .Plasmids are what found inside of the bacteria. Plasmids are important in this process of transformation because it has two essential features. The first is that it has a DNA sequence that helps plasmid replication. Plasmid replication is when genetic elements are in control within the host. The second is that the plasmid has a genetic
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More specifically‚ a previously prepared pGLO plasmid--which consisted of the gene to be cloned--was used to transform non-pathogenic bacteria. The pGLO plasmid contained a gene for the Green Fluorescent Protein (GFP) from a bioluminescent jellyfish and a gene for resistance to ampicillin‚ an antibiotic. Essentially‚ we wanted to determine the conditions of the bacteria that would glow. Our hypothesis was that the transformed solution with no plasmid DNA and ampicillin would produce no bacteria colonies
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In this experiment we transformed a strain of E. Coli bacteria without antibiotic resistance with plasmid DNA. This plasmid produces a fluorescent green glow under black light due to the gfp(green fluorescent protein) as well as antibiotic resistance. E. Coli cells will be plated on an agar medium‚ some with and some without the antibiotic ampicillin. Only bacterial cells that contain the plasmid will survive the ampicillin and produce the green glow. This experiment allowed us to observe the
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E. Coli. Chromosomal DNA of vibrio fischeri was first extracted and digested with restriction enzyme Sal I‚ then ligated with the vectors and transformed into the E. Coli cells. A few white colonies indicating the E. Coli cells took up the hybrid plasmids were observed on the plate but no glowing colonies were detected. The lux operon was not successfully cloned in this experiment. Introduction Vibrio Fischeri possesses a system called quorum sensing as a mean to express bioluminescence and communicate
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variability in bacteria. This experiment is based on the transformation mechanism of bacteria and gene regulation. The bacteria used for the experiment was Escherichia coli and the genes introduces for the transformation were: gfp and bla by a pGLO™ plasmid. After the insertion of the target genes and growing the bacteria on specialized LB media‚ it could be seen that the transformants were positive for the gene expression. The transformed E. coli on the media appeared fluorescent green under UV light
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miniprep‚ a DNA plasmid was isolated from the donor and transconjugant strains and FIGE electrophoresis was used to determine the size of the plasmid. The conjugation efficiency was found to be 16.25% and the plasmid DNA was approximately 97 kilobases long. The results show that the F’ plasmid was effectively transferred from the donor cells into the recipient cells via conjugation. Introduction:Bacterial conjugation is the unidirectional transfer of either genomic DNA or plasmid DNA from a donor
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Transformation in bacteria is the genotype alteration by the uptake of naked‚ foreign DNA from the environment. This concept of transformation was first discovered when Fred Griffith an experiment using mice and strains of pneumonia. Griffith concluded that a “principle” was transferred from heat-killed S strains to the R strains‚ which transformed them into deadly S strains. Oswald Avery later determined‚ through a series of experiments‚ that DNA was the “principle” that caused the R stains to become
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II:Total Genomic Extraction & Plasmid Extraction;Electrophoresis By:Chris Foster 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
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this experiment was to successfully ligate RFP into P1 and transform E. coli with P1-RFP plasmid‚ which contains the P1 promoter‚ RBS‚ and RFP gene. We tested several different ligation conditions and assessed how they each affected ligation efficiency. The two variables that we tested were: molar ratio of insert (RFP): vector (P1 plasmid)‚ as well as the effect of removing the stuffer from the digested plasmid. We predicted that the cells transformed without stuffer would have higher ligation efficiency
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