Recombinant DNA Technology: Results and Discussion

Recombinant DNA Technology
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Recombinant DNA Technology
Results and Discussion
Different samples of pBluescript plasmid inserted into lambda DNA were prepared in the first stage of the experiment. Seven plates were labeled, and each served a different purpose. Plate 1 served to produce the white colonies. Plate 1 produced the colonies because the recombinant pBluescript plasmid with lambda DNA was introduced to competent E.coli cells after being digested by HindIII, dephosphorylated and ligated. Plate 2 was a control sample to show the role of dephosphorylation in cloning as the plasmids did not undergo dephosphorylation. As expected, the plasmids underwent self-ligation, and there were fewer plasmids devoid of lambda DNA. This would also lead to less white colonies in plate 2.
Plate 3 also controlled for the role of dephosphorylation since there was no lambda inserted into the plasmid. Plate 4 served to control for the importance of ligase activity in cloning. In this plate, neither dephosphorylation of HindIII digested plasmid DNA was done nor the integration of the plasmid with lambda DNA. It was anticipated that the sample in plate four would undergo ligation of the plasmid DNA to produce a complete plasmid from the fragments. If this occurred, then the E.coli cells would be Lac+ because of the lack of the insert lambda DNA disruption. This is the phenotype expressed by the gene coding for Lac+. The presence of white colonies in plate four would be an indication that ligation did not occur or the process is insignificant in cloning. Plate 5 controlled for the competency of E.coli cells. It is expected that competent bacterial cells take up the pBluescript plasmid and this is the reason why normal pBluescript plasmid was added without dephosphorylation, ligation, or HindIII digestion. In plate 5, we expected a large number of white colonies since the cells were competent. In plate 6, control for any potential contamination was investigated because contamination largely affects the cloning results. This plate contained a sample with neither lambda DNA nor pBluescript plasmid and thus served as a negative control. Plate 7 was set to control for HindIII digestion activity. In the experiment, only those samples containing the pBluescript plasmid with HindIII were mixed with E.coli on plate 7. In the presence of HindIII, the plasmid DNA is digested to obtain DNA fragments, which if not ligated, they will not carry any functional DNA sequence. Therefore, in plate 7, it was not expected to have any blue or white colonies. According to the expectations, our group noted that no colonies were forming in both plate 6 and 7 and no possible contamination took place.
Plate 1 contained the highest percentage (51.72%) of the total number of white colonies in the experiment. Plates 2, 3,4,5,6, and seven did not contain any white colonies. Plate 1 was purposely set to show the number of colonies, which have been transformed using the plasmid with the insert DNA, or the cloned DNA. pBluescript plasmid, which was plated in the media, underwent ligation with the insert DNA, digestion with HindIII, and dephosphorylation with phosphatase enzymes. Adding plasmid DNA that...