Polymerase Chain Reaction Lab Report
The procedure was taken from “From Drosophila cDNA in E. coli plasmid to homologous human proteins” lab manual (4).
- Colony Picking: Two E. coli colonies were grown on agar plates and treated with ampicillin. They contained the plasmid with genes for ampicillin resistance and Drosophila cDNA sequence.
- Plasmid Isolation: We used the QuickLyse Miniprep Plasmid DNA purification systems to isolate the plasmid DNA. Indeed, the bacterial cells were removed from the liquid broth and were resuspended in the lysis solution. They were rinsed in a buffer before going through centrifugation.
- Polymerase Chain Reaction (PCR): The isolation done, the Drosophila cDNA located in the plasmid DNA was amplified. The PCR was used to determine the size of the cDNA inserted into the plasmid DNA. One tube (A) contained the plasmid DNA A with the PCR master mix, the other one (B) contained the plasmid DNAB with the PCR master mix, the last tube (negative control) contained PCR master mix with sterile water. A negative control was used to determine whether or not cross contamination had occurred.
- Agarose Gel Electrophoresis: The gel electrophoresis was used in order to determine and visualize and size the plasmid DNA and the PCR product. The gel electrophoresis was prepared using …show more content…
It is important to note that the cDNA sequence we analyzed was from a previous year (5-2-A_SP6_C1_Oct-1-2015.ab1) because our cDNA sequence data was poor and contained low peaks. We believed that the bad cDNA sequence was the result of contamination that may have occurred during the experiment. The Blast tool was used to find the mRNA that my cDNA sequence matched, then to determine what Drosophila protein the mRNA translates into. The ultimate goal of this step was to find if my protein had any homologous in humans and to determine if my protein was involved in any human
- Colony Picking: Two E. coli colonies were grown on agar plates and treated with ampicillin. They contained the plasmid with genes for ampicillin resistance and Drosophila cDNA sequence.
- Plasmid Isolation: We used the QuickLyse Miniprep Plasmid DNA purification systems to isolate the plasmid DNA. Indeed, the bacterial cells were removed from the liquid broth and were resuspended in the lysis solution. They were rinsed in a buffer before going through centrifugation.
- Polymerase Chain Reaction (PCR): The isolation done, the Drosophila cDNA located in the plasmid DNA was amplified. The PCR was used to determine the size of the cDNA inserted into the plasmid DNA. One tube (A) contained the plasmid DNA A with the PCR master mix, the other one (B) contained the plasmid DNAB with the PCR master mix, the last tube (negative control) contained PCR master mix with sterile water. A negative control was used to determine whether or not cross contamination had occurred.
- Agarose Gel Electrophoresis: The gel electrophoresis was used in order to determine and visualize and size the plasmid DNA and the PCR product. The gel electrophoresis was prepared using …show more content…
It is important to note that the cDNA sequence we analyzed was from a previous year (5-2-A_SP6_C1_Oct-1-2015.ab1) because our cDNA sequence data was poor and contained low peaks. We believed that the bad cDNA sequence was the result of contamination that may have occurred during the experiment. The Blast tool was used to find the mRNA that my cDNA sequence matched, then to determine what Drosophila protein the mRNA translates into. The ultimate goal of this step was to find if my protein had any homologous in humans and to determine if my protein was involved in any human