1 1. The structure of a human gene is given below, with boxes indicating exons and lines indicating introns. Assume complete splicing. Arow indicate position and direction (5'→ 3') of primers used for PCR. Sizes of each segment in kb are indicated below the diagram. Draw the expected results on the gel below for each experiment. Lane A: PCR of genomic DNA using primers 3 and 4. Lane B: PCR of cDNA using primers 3 and 4 Lane C: PCR of cDNA using primers 2 and 4 Lane D: PCR of cDNA using primers 1 and 4 2 3 2 kb 0.5 kb 2 kb 0.5 kb 1 kb 2.5 kb 1 kb 10 9 8 7 6 5 4 3 2 A B C D 4

1 1. The structure of a human gene is given below, with boxes indicating exons and lines indicating introns. Assume complete splicing. Arow indicate position and direction (5'→ 3') of primers used for PCR. Sizes of each segment in kb are indicated below the diagram. Draw the expected results on the gel below for each experiment. Lane A: PCR of genomic DNA using primers 3 and 4. Lane B: PCR of cDNA using primers 3 and 4 Lane C: PCR of cDNA using primers 2 and 4 Lane D: PCR of cDNA using primers 1 and 4 2 3 2 kb 0.5 kb 2 kb 0.5 kb 1 kb 2.5 kb 1 kb 10 9 8 7 6 5 4 3 2 A B C D 4

Biology: The Dynamic Science (MindTap Course List)

4th Edition

ISBN:9781305389892

Author:Peter J. Russell, Paul E. Hertz, Beverly McMillan

Publisher:Peter J. Russell, Paul E. Hertz, Beverly McMillan

Chapter19: Genomes And Proteomes

Section: Chapter Questions

Problem 1TYK

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1. What happens in the denaturing step of PCR? A. What happens during the annealing step of PCR? B. What happens during the extension step of PCR? C. What temperatures is associated with each PCR step? D. Why is it necessary to have a primer on each side of the DNA segment to be amplified? (Hint: draw it out to see what happens with only one primer). E. How did the Taq DNA polymerase acquire its name? F. Why are there nucleotides (A, T, G, C) in the master mix? What are the other components of the master mix and what are their functions?
1. You decide to perform dideoxy sequencing on a PCR product. You add the appropriate 32P- labeled primer, DNA polymerase, DNA template (the PCR product), buffer, DNTP mix, and a small amount of one of the four ddNTPs to four reaction tubes. You run the reactions in the thermal cycler, load each reaction into a separate lane of a polyacrylamide gel, and separate the products by gel electrophoresis. In the figure below, the lanes are labeled according to the ddNTP added. ddATP ddTTP ddCTP ddGTP - Lane 1 2 3 4 a) In lane 5, draw what you would expect to see if you prepared a reaction using a nucleotide mix containing only DATP, DTTP, dCIP, and dGTP. b) In lane 6, draw what you would expect to see if you prepared a reaction using a nucleotide mix containing only ddATP, DTTP, dCIP, dGTP. ||
9. Estimate the sizes of the bands, in each of the lanes for the gel attached below. Then, for each lane, match the fragments created with the set of restriction enzymes used in the digestion of 640 bp DNA.
Second part asks: Explain the roles two of those enzymes would play in obtaining the recombinant plasmids
19. You run a PCR of the above HBB sequence using the primers you designed. You receive a vector from a company that you want to clone your HBB PCR product into. Below is the vector: BamHI 0.5 kb EcoRI 1.0 kb 0.3 kb -Bglll plasmid A 0.2 kb BamHI 0.4 kb HindIII 1.0 kb EcoRI You want to cut this vector with BamHI and EcoRI and insert your PCR product into this vector. After cutting the vector with both enzymes simultaneously you run a gel. i) On the gel below draw the bands you expect to see after you cut this vector with these enzymes at the same time (only use one gel lane - the other one is going to be empty). (6 points) Ladder 5kb 3kb 2.5kb 2kb 1.5kb 1kb 0.5kb 0.3kb 0.2kb ii) Circle the band that represents the backbone that you will insert the PCR product into. (2 points) c) Once you have inserted the PCR product into the plasmid backbone from part C, how many bp will the new vector be (the total of the backbone and the PCR product together). Answer should be 2 significant figures.…
3A.Below are a few (of the many) possible errors that could adversely affect PCR or analysis of PCR products. Explain why these errors are bad and predict if the error would lead to no product or just a low yield. i) Primer and DNA volumes reversed (2.5 µl primer and 22.5 µl cheek DNA) ii) Student is homozygous for a mutation that deletes the four bases that would normally anneal to the last four bases at the 3´end of the primer iii) The PCR tube was not capped tightly (prior to the tube being placed in the thermal cycler) iv) While preparing a sample of the PTC PCR reaction to run on a gel, you intend to pipet up 8 µl of FD1 loading dye, but inadvertently depress the pipet plunger to the second stop instead of the first stop. How would this pipetting error affect your conclusions from the Flash Gel? Note: the error was made while preparing the sample with dye not while loading the gel.
1. What two restriction sites are you going to use to clone your PCR product into the pL4440 plasmid? What are their DNA sequence? 2. State the primer sequence you will use to amplify the F27C1.7 gene ready to be cloned into the pL4440 plasmid? 3. How would you go about cloning this amplified DNA into pL4440? Using your knowledge of cloning list 5 important aspects of the method.
1. The final amount of each primer required in a PCR reaction is 0.025 nanomol. If the total volume of the PCR reaction is equal to 100 µl and the stock concentration of each primer is equal to 10 µM. Calculate the volume of stock primer that needs to be added in order to ensure a primer amount of 0.025 nanomol. 2. A 250 ml phosphate buffer solution contains 204.12 µg KH2PO4 and 87.09 µg K2HPO4, respectively. Determine the molarities in micromolar (µM) of the abovementioned compounds in the phosphate buffer solution. [Mw KH2PO4 = 136.08 g/mol and Mw K2HPO4 = 174.18 g/mol] 3. You are required to use the pET 28b expression vector in a cloning experiment. If the vector is 5.368 kilobasepairs in length and the mass of the DNA that you have added represent 8.5 x 109 molecules. Determine the mass of vector DNA that you have added to your reaction? [1basepair ~ 660 Da or g/mol, Avogadro’s constant = 6.023 x 1023 molecules/mol]
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1. Primers come at a concentration of 200 pmol//ul (uM). You want the final concentration of your primers to be 0.2 uM. How would you dilute the primers in water so that you can add 1 ul in a PCR mix with a total volume of 50ul?
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