1) You want to clone the arg3 gene, which encodes an enzyme in the arginine biosynthetic pathway in Neurospora crassa. You have available the E. coli plasmid vector shown in part c below, a tube of Neurospora genomic DNA, a tube of Neurospora cDNA, and a method of introducing recombinant DNA samples into Neurospora.
a) Which would you prepare to try to rescue the arg3- mutant phenotype by transformation rescue? Circle one.
a) Genomic DNA library completely digested by your restriction enzyme of choice
b) A genomic DNA library partially digested by your restriction enzyme of choice
c) A cDNA library
b) Briefly explain your answer.
c) The plasmid below has an origin of replication (ori), a gene that confers resistance to streptomycin (strR), and the lacZ gene which converts X-gal to a blue compound. All of the sites for the restriction enzymes HpaII, EcoRI, HindIII, PstI, and SmaI are shown on the plasmid. Which restriction enzyme would you cut the vector with to make your library?
d) Using the source of DNA you chose in part a and the enzyme you chose in part c, list the steps you would follow to generate recombinant DNA molecules, get them into bacterial host cells (should these bacteria be resistant or sensitive to strep? should the bacteria have a functional lacZ gene?), identify those bacteria containing vector molecules with inserts, and then use transformation rescue to figure out which recombinant DNA molecules contain the arg3 gene.