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Cloning Brachyury from SW480 in pNEB193 plasmid - Essay Example

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Hence, all the cells were removed and stored them in preparation for the RNA extraction. Examinations and recordings were done to examine if the cell count and cell morphology were allowed before the step of…
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Cloning Brachyury from SW480 in pNEB193 plasmid
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Download file to see previous pages igure 2: The total RNA was extracted from SW480 cells by use of Norgen’s Total RNA Purification Kit, the samples were then denatured in rapid Formalin – free RNA loading buffer which had Formalin – Free RNA dye. They were then incubated for 5 minutes at a temperature of 700C. Lane number one was filled with RNA ladder 5 μL. The lanes from number 2 to 20 contained 10 μL of each of the class samples. The image capture was then done using GelDocEZ system.
3-Table 1: The concentration and purity of the total extracted RNA from the SW480 cells for the sample H was is shown in table 1. The resulting concentration of RNA was 82.88 ng/ul. An RQI of 7.3 indicated that the RNA quality was accepted. The ratio (28S/18S) was 0.93 though the recommended ratio is 2
The plasmid preparation experiment was undertaken before the start of the RNA extraction. The purpose of this experiment was to purify enough linearized phosphatise treated pNEB183 plasmid to be utilised in the ligation reaction. This purification was attained through several steps that started with the purification of the inoculated plasmid from E.coli culture in LB/ampilicin broth. Using Qubit analysis, the concentration of the purified plasmid was calculated to be 5.1 ul. The EcoR1 enzyme was then utilised to digest the circular plasmid into a linear plasmid which was then treated using alkaline phosphatise enzyme to remove the 5’ phosphate group and hinder self – ligation. The sample was then loaded on 0.8% agarose gel so as to visualize and purify the linearized plasmid from the gel by use of purification method as shown in figure 5:
In figure 5, two bands were clearly seen in the second lane. The first band was an uncut plasmid. The second band was a linear plasmid. Adequate preparation took place as the linear plasmid could migrate longer on the gel. The second band was bright and shiny. It was 2797 bps in length. It also contained ...ul/mg concentration of plasmid. Using x tracta Gel Extraction ...Download file to see next pagesRead More
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