Plasmid Isolation Lab Report

Our goal within the plasmid isolation lab was to isolate the bacterial plasmid without any damage to the DNA. The formation of the clear lysate was essential because it allowed us to isolate the bacterial cells from removed media and later expose the DNA. Resuspension solution helped to resuspend the cells, after centrifugation, and prepare the cells for lysis solution. The lysis solution functions to degrade the cell membrane and spill its components. Then the Alkaline Protease functioned as the inhibitor of any DNases and denatured dsDNA to ssDNA shortly, by disturbing H-bonds between bases. Finally, the Neutralizing Solution was helpful into stabilizing the pH, from a basic solution formed by the lysis and protease solution.
The DNA was
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RNA was used as the template for reverse transcriptase, which would give rise to the cDNA that would later be used as the template for PCR. However, some tubes like the –RT and –RNA, which served to be our negative controls, do not contain RT or RNA, respectively. Random Primers attach to the RNA molecules, giving an indication of where the RT should bind. 5x Reaction buffer was used to maintain the pH of solution and maximizing the efficiency of the product production; while MgCl2 aids in the activity of the enzyme. In order for the reverse transcriptase to attach the appropriate base pairs for the cDNA, we had to add dNTPs. RNasin helps by inhibiting any RNases that may degrade our RNA templates. Reverse Transcriptase (RT) is the enzyme that uses RNA as a template and transcribe DNA.
Within the preparation of the RNA tubes, first incubation, time of 5 minutes at 70° C was required to remove any secondary structures that may be present. A second incubation (25° C for 5 minutes) was needed to allow the primer to anneal to template and to the enzyme, after combining the appropriate tube together. Third incubation at 42° for one hour allowed enough time for transcription to occur. Finally, the fourth was used to inactivate the enzyme and stop transcription, which was run for 15 minutes at 70°
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Since reverse transcription is non-specific to RNA molecules, there is the possibility of having mRNA that code for proteins other than Actin. To solve this problem, there were specific primers that were used in PCR, primers 1 and 2 (forward and reverse, respectively); these primers are specific for gene of interest. The primers are Actin forward (1) and Actin reverse (2). During preparation of the reaction, most of the time the reagents are left on ice, this is to prevent the primers and enzyme to anneal to the templates before reaction. Once the master mix was ready, 19 µL was taken and added to three new tubes, and one kind of template was added to one specific tube. Therefore, in the end the three tube contained only one template. This was done to test if there was any product that would form in the negative controls compared to the positive control. One the reaction tubes were ready, we add them into the thermocycler. The thermocycler is where PCR will occur. It consists of several cycles, about 35 cycles that amplify a single DNA sequence to over 2¬¬¬35 copies. Once the DNA is amplified, it can be analyzed on a gel. My products in the +RT tube were analyzed; as expected I found a bright band around the 1100 bp, indicating that my amplified sequence is as expected. However, in the –RT, a dim band was found around the 1300 bp, indicating a possible genomic DNA contamination. Finally, the –RNA did not show any

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