{"id":4618,"date":"2020-05-18T00:00:00","date_gmt":"2020-05-18T00:00:00","guid":{"rendered":""},"modified":"2020-05-18T00:00:00","modified_gmt":"2020-05-18T00:00:00","slug":"subcloning-experiment-transfer-of-a-dna-fragment-from-pmb-to-puc19","status":"publish","type":"post","link":"https:\/\/www.ukessays.com\/essays\/biology\/subcloning-experiment-transfer-of-a-dna-fragment-from-pmb-to-puc19.php","title":{"rendered":"Subcloning Experiment: Transfer of a DNA Fragment from pMB to Puc19"},"content":{"rendered":"

\n\t\t\t\tSubcloning of the PstI fragment from pMB into pUC19\n\t\t\t<\/p>\n

\n\t\t\t\t1. introduction<\/strong>\n\t\t\t<\/p>\n

\n\t\t\t\t      DNA should never be treated of considered as a static molecule since those molecules have a significant dynamic movement. It is constantly changing, and one mechanism by which this change is brought about is recombination.\n\t\t\t<\/p>\n

\n\t\t\t\t     Recombination is the ability to transfer a specific gene from one organism to another resulting in offspring differ from either parent. It is a DNA re-arrangement between\n\t\t\t<\/p>\n

\n\t\t\t\ttwo molecules of DNA having homologous sequences. The main objective of doing recombinant DNA technology is often to create a beneficial product or commercial ones.\n\t\t\t<\/p>\n

\n\t\t\t\t      Recombination provides the tools through which genetics information is reformed. This, of course, provides an evolutionary advantage to eliminate the unwanted\n\t\t\t<\/p>\n

\n\t\t\t\tmutations and to maintain the spreading of the desired mutations. Moreover, it has a significant role in conferring each individual a unique group of genetic information. Recombination must be taken place between precisely corresponding sequences to ensure no loss or addition for any base pair.\n\t\t\t<\/p>\n

\n\t\t\t\tAfter the recombination has been done, in order to identify the recombinant vector from non-recombinant ones, two techniques must be used (Bolivar et al<\/em>, 1977). First, growing the E. coli on the antibiotic-containing medium, which contains ampicillin. This one used to distinguish the recombinant bacteria from the non-recombinants. That can be determined easily since recombinant bacteria carry plasmid that has the ampicillin-resistance gene (AmpR), and in which can grow on ampicillin based medium, however, the non-recombinants cannot grow on the growth media since they have no resistance to the ampicillin (Smalla, Jechalke and Top, 2015). The problem, not all the plasmids transformed into cells may contain the inserted gene. Some of the cells do have the plasmid but without the inserted gene.\n\t\t\t<\/p>\n

\n\t\t\t\tThe second technique is the LacZ <\/em>a-complementation (Fig1), which is a screening technique that allows the detection and distinguishes recombinants bacteria with insert DNA fragment from the one with only the plasmid but without the inserted gene by using blue\/white selection to identify recombinant plasmid clones (Tolmachov, 2009).\n\t\t\t<\/p>\n

\n\t\t\t\t \u03b2-galactosidase is a protein produced by E. coli strain that carries the lacZ gene. In its active state, if bacteria are grown on an agar plate that has a substrate known as X-gal<\/a>. when \u03b2-galactosidase is produced, X-gal is hydrolyzed(Tolmachov, 2009). Consequently, they produce an insoluble blue pigment. However, when Insertion of foreign DNA from pMB into the plasmid at the MCS region, which is located within the lacZ\u03b1 sequence <\/em>(which can be cut by restriction enzymes),that would cause insertional inactivation(Smalla, Jechalke and Top, 2015). That would create a non-functional \u03b2-galactosidase enzyme, \n\t\t\t<\/p>\n

\n\t\t\t\tthereby it will stop the gene that produces \u03b1-peptide from working. Consequently, in cells that contain the plasmid with an insert in LacZ\u03b1 <\/em>sequence,<\/em> no functional \u03b2-galactosidase will form (Smalla, Jechalke and Top, 2015).\n\t\t\t<\/p>\n

\n\t\t\t\tTherefore, The E.coli that has plasmid with no insertion appears blue in color since LacZ is functioning while the recombinant ones with insertion foreign DNA appear white<\/a>. Consequently, it\u2019s easy to pick the desired recombinant colonies from the culture.\n\t\t\t<\/p>\n

\n\t\t\t\t\"\" \n\t\t\t<\/p>\n

\n\t\t\t\t <\/strong>\n\t\t\t<\/p>\n

\n\t\t\t\t <\/strong>\n\t\t\t<\/p>\n

\n\t\t\t\t <\/strong>\n\t\t\t<\/p>\n

\n\t\t\t\t <\/strong>\n\t\t\t<\/p>\n

\n\t\t\t\t <\/strong>\n\t\t\t<\/p>\n

\n\t\t\t\t1.1 Aim of the experiment<\/strong>\n\t\t\t<\/p>\n

\n\t\t\t\tSubcloning experiment: transfer a DNA fragment from pMB to Puc19. Then detect the bacterial clones carrying the recombinant plasmid by using the insertional activation system.  Lastly, successful subcloning demonstrated by using restriction digest to confirm that the bacterial clones do contain recombinant plasmids.\n\t\t\t<\/p>\n

\n\t\t\t\t\"\"2. Results:<\/strong>\n\t\t\t<\/p>\n