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I need help creating a thesis and an outline on Cloning Brachyury from SW480 in pNEB193 Plasmid. Prepare this assignment according to the guidelines found in the APA Style Guide. An abstract is requir
I need help creating a thesis and an outline on Cloning Brachyury from SW480 in pNEB193 Plasmid. Prepare this assignment according to the guidelines found in the APA Style Guide. An abstract is required. For the quality of the RNA that was determined in the experiment to be analyzed, denaturing agarose gel electrophoresis was utilized to view the band's 18s and 28s. The two kinds of RNA are utilized to evaluate the quality of mRNA that was selected in the experiment.
This step which involved making DNA from the RNA that was determined in the first step was very important. The RNA templates which were used at the start of the experiment were taken from the samples. Using a SuperScript III system and the primers Oligo (dT)20, each student synthesized a cDNA from 741ng of RNA. The concentration of the synthesized cDNA was measured using a Nanodrop. The results are shown in Table 2.
The concentration of my sample F was 302.3ng/μL. This was a very low concentration in comparison with the average of the class. Additionally, the standard deviation was 131.2. This meant that all the data obtained tended to close to the mean. Hence, the class did quite well in the experiment.
The explanation for this step was to test whether the cDNA was appropriate for the PCR. Due to this, two primers were used to amplify the β – actin. This produced 353bp of the PCR product. Hence, the end product of the PCR was made up of the total cDNA length and there was no more amplification for the genomic DNA in the sample.
On the evaluation of the agarose gel that had 0.8 % concentration by use of image lab, it was discovered that lane 1 had 3 faint bands that were 1325 and 1400bp at the top by approximation. This seemed to be a good result as Brachyury was amplified by primers from cDNA. Nonetheless, a second band was observed. This indicated that there was more than one template that could be connected to another kind of Brachyury which was in the established cDNA from the previous step. Band number 3 which was quite faint was the primer – dimer. A very bright single band (333bp) could be observed in lane 2 where β – actin was loaded onto the gel. This signified that cDNA was appropriate for PCR amplification.
