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Cloning Brachyury from SW480 in pNEB193 plasmid - Essay Example

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It was under the supervision of an experienced demonstrator. Each and every student had the same materials and also followed a similar protocol for each step of the practical. Hence, it is most…
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Cloning Brachyury from SW480 in pNEB193 plasmid
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Download file to see previous pages This concentration permitted me to proceed on to the next step of the experiment as it had more than 500 ng of RNA. However, it was noted that some students obtained very low concentrations of 15 ng/ μL compared to the class average of 169.3 ng/ μL. If the class results are analyzed, it can be noted that the standard deviation of this experiment is 151, hence, the range of results is (169 +/− 151) 18 – 320. Table 1 shows that sample F concentration. Nevertheless, samples H, L for example are much higher than the range of the results. The key point of the experiment which determined if the experiment could go on or not was the quality of RNA. That is depicted in figure1: gel denaturation of RNA, the ratio between 28S and 18S RNA is 1.3 – 2.5; many of the students had this value while some were not due to sample degradation or they did not load their samples in the appropriate wells.
3 – The second experiment was the cDNA synthesis where RT – PCR (SuperScript III RT) was used to synthesis it from RNA which was determined by the students as B, D, E, G K O and P. Every student began with a concentration of 6.16 ng. After the synthesis of cDNA, the concentration of the the whole sample was measured by the demonstrator using a Nanodrop. The data of the whole class was very close to the average 747 .2 ng/ μL (table 2). The standard deviation was 131.2 which were very small. Thus, almost all students did the experiment correctly as the results of the class data were in the range of 547 – 631 ng/ μL. On my part, the sample obtained was 302 ng/ μL which also was in the range. This allowed all students to go on to the next experiment.
4 – The next step of the experiment involved the use of the PCR to amplify the full length of Brachyury and human β – actin from the cDNA that was obtained. Using 0.8% agarose gel electrophoresis, the products of the PCR were analyzed. The PCR in my group worked and had ...Download file to see next pagesRead More
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