Discussion & Conclusion
RNA isolation using TRIzol provide a fast and clean isolation. TRIzol is an ideal single-step reagent for the isolation of RNA because of the high accuracy by maintaining the RNA integration while disrupting the cells and make the cell components soluble (Simms et al., 1993). The RNA remain in the aqueous phase, which can be precipitated by isopropanol. By quantifying, as seen in Table 1., the A260/A280 ratios of 1.93 and 1.99 suggest that the isolated RNAs are pure as the ratio is approximately 2.0. However, Nano readings are not 100% accurate as the values provided are different every time, despite using the same sample. Any values below 2.0 will suggest that there are contaminations, either by proteins or DNA molecules…show more content… However, it does not necessarily mean that the E. coli bacterial cells took up the recombinant vector. Although The E. coli bacterial cells are rendered competent by Ca2+ and able to take up foreign DNA, the foreign DNA taken up might be recombinant vectors, self-recombined plasmids or self-recombined DNA fragments. This is because both the cut plasmid and the cut RT‐PCR fragment ligation have the tendency to undergo self-recombination. There might be partial digestion of plasmid pET11a, which leads to restriction sites that are not cleaved and result in the intact plasmid. Both the self-recombined plasmids and the recombinant vectors will have ampicillin resistance property and thus, it is inaccurate to assume that all the colonies grown have taken up the recombinant vectors (Hanahan, 1983). However, the self-ligation can be prevented if phosphatase is added to remove the phosphate group in the nicked plasmid. This will allow the phosphorylated DNA insert to enter the dephosphorylated DNA plasmids. In this case, since the phosphatase is not added, DNA ligation and gel electrophoresis are required to confirm the presence of the…show more content… 3., are quite constant but the uncut and single cut plasmids were not observed, along Lanes 1 and 2. It is crucial to know the uncut pasmid as it serves as a positive control to check the plasmid which did not undergo DNA digestion. However, there are 3 possible results, with Lane 10, being the most accurate and sharp band. It is proved to be accurate as there are 2 bands observed along the lane. The DNA band, at about 1,000bp shows the possible presence of the correct insert.
For Plasmid 1 that undergoes DNA sequencing, the insert is located 5282nd nucleotide to 6337th nucleotide in the correct orientation because of the two different enzymes used in DNA isolation. This is observed by comparing the DNA sequences from 4 different BLAST results from pET11a forward primer, pET11a reverse primer, LDHA 430 and 570. Abovementioned, the pET11a forward and reverse primers flank both the plasmid and insert sequences while the LDHA 430 and 570 primers flank inside the insert