Agro/ANSC/Biol/Hort/Gene 305 Fall, 2016 Homework #4 (Due back on Dec 2, 2016) Name: Section : Q1. Describe the important features of cloning vectors. Explain the purpose of selectable marker genes in cloning experiments.

Q2. In a stepwise manner describe the steps in cloning a gene. Your answer should also include how you would identify a bacterial colony that contains your gene of interest.

1 Q3. Some vectors used in cloning experiments contain bacterial promoters that are adjacent to unique cloning sites. This makes it possible to insert a gene sequence next to the bacterial promoter and express the gene in bacterial cells. These are called expression vectors. If you wanted to express a eukaryotic gene in bacterial cells, would you clone a genomic DNA or cDNA into the expression vector? Explain your choice. What other manipulations have to be done to ensure that the expression vector will allow the translation of the eukaryotic mRNA in the bacterial cells?

Q4. Starting with a sample of RNA that contains the mRNA for the -globin gene, explain how you would create many copies of the -globin cDNA using reverse transcriptase PCR.

Q5. In Southern, Northern and Western blotting, what is the purpose of electrophoresis?

2 Q6. To produce transgenic plants, plant tissue is exposed to Agrobacterium tumefaciens containing a gene of interest, and then grown in media containing kanamycin, carbenicillin and plant growth hormones. Explore the purpose of each of the three agents. What would happen if you left out kanamycin?

Q7. What is the strategy for expressing human genes in a domestic animal’s milk?

3