Question

Tom is a molecular biology graduate student who needs to isolate a gene from the pathogenic bacteria Salmonella typhi, which he is studying. He wiselydecides to use PCR to amplify the gene before trying to purify it. The gene has the general structure shown below:

~1000 Base Pairs ! ! 5’-ATGGCTTACGTG-----------CCATAGGGCTAA-3’ ! ! 3’-TACCGAATGCAC-----------GGTATCCCGATT-5’

The gene is about 1000 nucleotide base pairs long, but only the sequences at the ends of the double strand are shown. Use the information from the video, what you learned about base pairing in class, and the fact that PCR always proceeds along the parent DNA strand the 3’ to 5’ direction. Design two, 6-nucleotide long, PCR primers for Tom. When you have these primers designed, proceed to the “Exercises and videos” link. You will be prompted for important information in drop-down menus. Answer each question and click continue to see a first-person performance of a PCR reaction.

Question #1 (These will be multiple choice and are automatically graded) a) What different components will do you want to add to your PCR?

b) Which primers will you use?

c) How many PCR cycles should you set the thermocycler for to obtain 128 TOTAL strands of DNA at the end of the reaction (this includes the template strands that you started with)?

d) To analyze the PCR you need to make a DNA gel. What do you want to add to make the gel?