Quiz 6: Molecular Cloning PDF

UST / BIOL3321: Genetics My Grade: ____________ / 100 Formative Assessment My Time: ___________ min Quiz 6: Molecular Cloning True...

UST / BIOL3321: Genetics My Grade: ____________ / 100 Formative Assessment My Time: ___________ min Quiz 6: Molecular Cloning True or False? (3 points each = 18) 1. __F____ In blue-white screening, one should always select the blue colonies. 2. __F____ Colonies are blue due to the disruption of the lacZ gene during cloning. 3. __T____ The DNA fragment or gene of interest should always be inserted into the lacZ gene. 4. __T____ Xgal is the name of a lactose analog that when cleaved, produces blue coloration of the colony. 5. __T____ Restriction (digestion) will be used both for the cloning process and to check the orientation of the insert. 6. __F____ The same restriction enzyme should be used for cloning and to check the orientation of the insert. Multiple Choice (6 points each = 42) 1. What is the primary function of a restriction enzyme in molecular cloning? A) Amplifying DNA fragments B) Cutting DNA at specific sequences C) Joining DNA fragments D) Transcribing DNA into RNA 2. Which of the following is typically used as a vector in bacterial cloning? A) Plasmid B) Chromosome C) Virus D) Mitochondrion 3. What is the process called where a foreign DNA fragment is inserted into a bacterium? A) Ligation B) Restriction digestion C) Transformation D) Transcription 4. What technique is used to identify bacterial colonies containing a specific construct using a selectable marker? A) Gel electrophoresis B) PCR C) Southern blotting D) Blue-white screening 5. Which of the following enzyme is responsible for making a DNA copy from RNA? A) Reverse transcriptase B) DNA polymerase C) RNA pol I D) RNA pol II 1 6. Which of the following statements is false? A) The cells which take up recombinant plasmids are antibiotic resistant B) The cells with an empty vector are antibiotic resistant C) Plasmid replication is independent of cell’s division D) The cells containing a recombinant plasmid are called transformed cells 7. Which of the following statements is true? A) The cells with no vector are antibiotic resistant B) The bacterial cells that acquire a recombinant plasmid are blue C) Xgal is a compound that makes bacteria resistant to an antibiotic D) The bacterial cells that acquire a recombinant plasmid are white Molecular Cloning (40 points) 1. (10 pts) List the steps of molecular cloning. Insert preparation Vector preparation Ligation Transformation Colony screening 2. (30 pts) The sequence below (rizz) should be cloned into the vector shown below. Distances between restriction sites are indicated in bp (for example, ther are 80 bp between restriction sites B and A at the top of the image). The full plasmid is 5,000 bp. a. (10 pts) Select an enzyme to clone rizz into the vector. Explain why you selected that enzyme. B is the best enzyme to clone rizz into the vector because of two reasons: It cuts upstream and downstream of the gene of interest (but not in the gene of interest). It cuts the vector only once, in the lacZ gene. 2 b. (20 pts) Can restriction enzyme C be used to test orientation of the vector? Support your answer by giving resulting fragment sizes. Restriction enzyme C can be used to test orientation of the insert. If the insert is in the correct (forward) orientation, the digestion would produce two fragments, one of 230 bp and the other of 5,650 bp. If the insert is in the incorrect (reverse) orientation, the digestion would produce two fragments, one of 650 bp and the other of 5,230 bp. 3