Q1 A research laboratory identified a gene X of medicinal valuein a plant species. You are given a small fragment of DNAcontaining gene X and the cloning vector pZoom. Maps of the 4 kbcloning vector pZoom and the 10 kb plant DNA fragment are shown inFigure 1 and 2, respectively. The PCR primer pairs (shown in Figure1) F1/R1, F2/R2 and F3/R3 amplify fragments of 0.1, 0.6 and 0.8 kb,respectively. The antibiotic resistance gene A and gene B code forchloramphenicol and streptomycin resistance, respectively. Thethree restriction enzymes that cut this vector are BamHI, XbaI andHindIII represented on the map as enzymes I, II and III,respectively. You are given purified DNA of both the vector and thegene X containing DNA fragment at 0.2 µg/ul. Create a recombinantplasmid containing the complete gene X in the provided cloningvector pZoom. a. Design restriction digestion reactionsusing appropriate enzymes in such a way that you get a finalconcentration of 50 ng/µl for the digested vector and for the plantDNA (insert DNA) in the reaction. Your digestion should include allcomponents in a 20 µL reaction. All enzymes are supplied with aconcentration of 10 units/µL; you may use 1 µL of the enzyme ineach reaction. Buffers for each enzyme are available as 10 timesconcentrated (10X) stocks. b. Generate three ligation reactionswith 1:1, 2:1 and 3:1 molar ratios of the insert and the vectorDNA. Your ligation should include all components in a 30 µLreaction. Keep the vector amount fixed at 100 ng per ligationreaction. You are provided with 10 x ligase buffer and DNA ligase(0.5 U/µL) to set up your ligations. c. Develop a strategy toselect transformants and a quick method to screen recombinants;show your screening method using a figure. Predict the expectedresults with an explanation. d. Devise a restriction analysismethod to confirm the desired recombinant; use a single mostappropriate enzyme. Calculate the expected sizes of the restrictionfragments from each, the vector and the desired recombinant. e.Devise a PCR strategy to confirm the desired recombinant. Calculatethe expected sizes of the PCR fragments from the desiredrecombinant in a multiplex PCR including all three primer pairs.How will these sizes differ from the same multiplex PCR if thetemplate is vector DNA instead of the recombinant plasmid?