Title: The purpose of the experiment is to analyze Drosophila melanogaster cDNA Sequences in order to find homologous proteins in humans of which can lead to a model for human disease. Introduction: Drosophila melanogaster, the common fruit fly, is a very advantageous organism to work with in the laboratory, for it is a very useful model for human disease. Much is known about these organisms since have been studied for over 100 years. Drosophila are very easy to culture and breed due to their small size and short lifespan of thirty days (NIH). They are easy hosts for conducting drug testing and, most importantly to this experiment, have a high homolgy of DNA sequence and conserved physiology when compared to humans (LAB BOOK). Proteins …show more content…
Selection of E. coli colonies for growth (Week 1) Two colonies (A and B) of E. coli which took up plasmids with Drosophila cDNA were picked and used to inoculate media. The E. coli cells act as a host organism to store plasmids containing Drosophila cDNA (PAPER). The media was left to grow, and produce a culture of bacteria with identical genomes, at 37oC overnight. The E. coli not only contained the Drosophila cDNA, but also genes which encode for ampicillin resistance in the cell. Ensuring that colonies were ampicillin resistant also ensures that only E. coli cells with the plasmid will grow. Plasmid Isolation and Polymerase Chain Reaction (PCR) (Week 2) Carefully using aseptic techniques, the plasmid DNA was extracted from the E. coli using QuickLyse Miniprep. In this method of isolation, the E. coli cells were immersed in a lysis solution, which released the plasmid DNA from the E. coli cells to be filtered in the spin column. The plasmid DNA was then washed with buffers to remove any …show more content…
Towards the top of the image shows the tiny compartments that each mixture was loaded into. The mixtures migrated downward toward the positive end of the gel. Plasmid B and PCR B do not appear in this image, signifying an error in the experiment. This error could be due to contamination during the plasmid extraction and/or PCR. The plasmids from sample B were still sent for sequencing, despite the error. Plasmids and PCR product from sample A are rather large (>2000 bp). The further they travel toward the positive end, the smaller the plasmids. The trace file analyzed was from plasmid sample A since plasmid sample B yielded no plasmid sequence when sent for sequencing. The request ID number recorded was 17HSTX1N014. The only protein domain found in the Drosophila protein was the S1_like Superfamily. The request ID number recorded was 17JN19JP01R. Discussion: Plasmid sample B was unable to be sequenced when sent to the Nucleic Acid Facility. It was also not seen on the agarose gel in the gel electrophoresis step. Error may have occurred when plasmid B did not extract from the E. coli properly. This error was likely the result of contamination during the plasmid extraction, which is a very sensitive process. Contamination could have occurred by not replacing the tip of the pipette during each