site (A… Show more The plasmid diagrammed below contains several unique restriction sites (E,X, and S); a site (A) present twice in the plasmid; a gene that confers resistance to ampicillin (AmpR); a gene that confers resistance to kanamycin (KanR); and an origin of replication (Ori). Note: The black triangles represent the positions of the cleaved phosphodiester linkage inside the restriction sites. 1) The plasmid (3500 bp in length) is digested with EcoRI and XbaI. We assume that the loss of nucleotides located between the two restriction sites is negligible. a) A fragment of DNA (F, 500 bp in length) was obtained by digestion of genomic DNA with the restriction enzymes EcoRI and SpeI. The cleaved plasmid and cleaved fragment F are mixed together, and then treated with DNA ligase. Bacteria, unable to grow in the presence of kanamycin and ampicillin, were transformed with the ligation mixture. Transformed bacteria are plated either on dishes containing ampicillin or on dishes containing kanamycin. Do you expect any colonies on the plates? Explain (4 pts) b) In a different experiment, you create a modified version of the plasmid. You digest the original plasmid with restriction enzyme A, and purify the longer piece of DNA resulting from the digestion. The purified long piece of DNA is then treated with ligase. Then, bacteria unable to grow in the presence of kanamycin and ampicillin are transformed with the ligation mixture. Due to some technical problem, the purified piece of DNA is contaminated with copies of the original plasmid. Therefore, you decide to transform the bacteria with a mixture of the original and the modified plasmid. Describe how you would quickly select bacteria transformed by the modified plasmid without extracting and sequencing the plasmid from transformed bacteria. (4 pts) 2) Assuming that you got bacterial colonies in the experiment described in part 1a. You purify the plasmids from one of these colonies and submit the plasmid to the following restriction digestion: Digestion #1: enzymes XbaI and SpeI Digestion #2: enzyme A How many DNA fragments do you recover after digestion #1 and after digestion #2? Explain your answers and when possible indicate the size of the DNA fragments. (4 pts) • Show less