(Solved): 2. You are using the pUC18 vector (again...), this time to clone a PCR fragment that you have ampli ...

2. You are using the pUC18 vector (again...), this time to clone a PCR fragment that you have amplified from human cDNA. The cDNA is YFG (Your Favorite Gene). You cut pUC18 with the restriction enzyme SmaI, which leaves blunt ends into which you can ligate the bluntended PCR fragment. Look at the diagram at right to see the restriction enzyme recognition sites available in the pUC18 Multiple Cloning Site (MCS) and the order of these sites on the vector. Note that RE recognition sites in the MCS are unique - they do not occur anywhere else on the pUC18 vector plasmid. You intend to use the lac promoter (Plac) present in the pUC18 vector to drive expression of the YFG cDNA. Again, you can look at the map to identify the direction that the promoter will drive transcription. Your PCR fragment has the following map: pUC18 has a multiple cloning site within the lacZ $Z$ alphafragment. Inserts cloned into this site disrupt beta -galactosidase (lacZ) activity and give rise to white colonies on X-GaVIPTG plates. The plasmid encode resistance to the antbiofic ampicillin. a. Draw a map of the plasmid with a CORRECTLY ORIENTED INSERT that you hope to obtain after ligation. Show the RE sites KpnI, and Sall contained in the vector and the insert. Also show the location of Plac, and indicate the direction that transcription will proceed from the Plac promoter. b. Draw a map of the plasmid with an INCORRECTLY ORIENTED INSERT that you will also obtain after ligation Show the RE sites $K$ pnl, and Sall contained in the vector and the insert. Also show the location of Plac, and indicate the direction that transcription will procecd from the Plac promoter. c. You have ligated the insen into the Smal cut vector, you have transformed it into lacZ- E. coli and plated on appropriate medium, and you have selected sevenal white colonies to analyze by preparing plasmid DNA and performing restriction enzyme digentions on it. If you cut your new clones with restriction cityme Saln, what fragments will you observe when your cDNA has been cloned in the CORRECT ORIENTATION? d. You decide to use restriction enryme $K_{pnl}$ to screce for your correctly otiented clones. At right is the gel you run affer preparing plaumid DNA from 8 white colonies and digesting if with Kppl. Which lane(0) contain the clone you want (correctly orienied $?G$ cDNA)?