PRESCOTT'S MICROBIO W/PROCTORIO
11th Edition
ISBN: 9781264731060
Author: WILLEY
Publisher: MCG
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Chapter 17.2, Problem 4CC
Why is it possible to visualize a PCR product on an agarose gel even if the template genome is present at such a low concentration that it cannot be seen?
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You wish to amplify a segment of DNA from a plasmid template by PCR with the use of the following primers: 5’-GGATCGATGCTCGCGA3' and 5' -AGGATCGGGTCGCGAG-3'. Despite repeated attempts, you fail to observe a PCR product of the expected length after electrophoresis on an agarose gel. Instead, you observe a bright smear on the gel with an approximate length of 25 to 30 base pairs. Explain these results.
Our PCR samples already contain loading dye, but sometimes this isn’t the case. If your samples didn’t already contain dye and you wanted to load your PCR sample onto an agarose gel, you’d need to add loading dye to the proper concentration. There is a 6X loading dye available for use; how many µl of this loading dye will you add to 10 µl of your sample so that it is at a 1X working concentration? Show your work.
In relation to the use of restriction enzymes in recombinant DNA technology, answer the following:
You have accidentally torn the labels off two tubes (tube A and tube B), each containing a different plasmid, now you do not know which plasmid is in which tube. Fortunately, you have restriction maps for both plasmids, shown in Figure below. You have the opportunity to test just one sample from one of your tubes. By utilizing agarose gel electrophoresis technique, which restriction enzyme OR combination of restriction enzymes would you use in this experiment to determine which plasmid is found in which tube?. (Hint: if you use Hind III restriction enzyme you are going to get ONE single fragment with a molecular size of → 0.5+0.3+0.2+0.4+1+1 = 3.4 kb).
Chapter 17 Solutions
PRESCOTT'S MICROBIO W/PROCTORIO
Ch. 17.1 - Examine the uncut piece of DNA shown in the upper...Ch. 17.1 - Which of the above enzymes yield blunt ends? Which...Ch. 17.1 - Prob. 3MICh. 17.1 - What would you conclude if you obtained only blue...Ch. 17.1 - Why must introns be removed from eukaryotic DNA...Ch. 17.1 - Which plasmid is a shuttle vector? Why?Ch. 17.1 - In what ways does the BAC shown here differ from...Ch. 17.1 - Describe restriction enzymes, sticky ends, and...Ch. 17.1 - What is cDNA? Why is it necessary to generate cDNA...Ch. 17.1 - Prob. 3CC
Ch. 17.1 - Prob. 4CCCh. 17.1 - Prob. 5CCCh. 17.2 - Why, after three cycles, are the vast majority of...Ch. 17.2 - Briefly describe the polymerase chain reaction....Ch. 17.2 - Why is PCR used to detect infectious agents that...Ch. 17.2 - How would you use PCR to measure the concentration...Ch. 17.2 - Why is it possible to visualize a PCR product on...Ch. 17.2 - Prob. 5CCCh. 17.3 - Why are long fragments (e.g., 20,000 bp) of...Ch. 17.4 - What special considerations are necessary if one...Ch. 17.4 - Prob. 1CCCh. 17.4 - Prob. 2CCCh. 17.4 - Prob. 3CCCh. 17.4 - You are studying chemotaxis proteins in a newly...Ch. 17.5 - Prob. 1MICh. 17.5 - Prob. 1CCCh. 17.5 - Prob. 2CCCh. 17 - Which of the DNA molecules shown are recombinant?Ch. 17 - Prob. 1RCCh. 17 - Prob. 2RCCh. 17 - Prob. 3RCCh. 17 - Prob. 4RCCh. 17 - Prob. 5RCCh. 17 - Prob. 6RCCh. 17 - Prob. 1ALCh. 17 - Prob. 2ALCh. 17 - Suppose you transformed a plasmid vector carrying...Ch. 17 - You are interested in the activity and regulation...Ch. 17 - Prob. 5ALCh. 17 - Prob. 6ALCh. 17 - Prob. 7AL
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- Why Touch-down PCR technique is better than the Gradient PCR technique? Also, from those techniques which technique produces the pure product?arrow_forwardBelow are gel electrophoresis results for initial and nested PCR of the Shrimp Plant. Use the results to answer these questions: a) Why does the Arabidopsis control generate two PCR bands, but the plasmid control only generates one PCR band? b) Did the negative control generate a PCR product? If so, what are the implications of this for the experiment? c) Would you use this nested PCR product from Shrimp Plant for cloning? Briefly explain your answerarrow_forwardlet three loci be X,Y,Z. three pairs of primes can be used to amplify these loci in multiplex PCR. the resulting amplicons(from different loci) are of the equal size. will this scenario be acceptable in STR typing? why or why not?arrow_forward
- A successful PCR experiment often depends on designing the correct primers. In particular, the T m for each primer should be approximately the same. What is the basis of this requirement?arrow_forwardA) What are the three main steps involved in PCR? Include temperatures and descriptions of each step. B) Explain why RFLP can produce many bands on an electrophoresis gel and PCR (one set of primers) will only produce one or two bands on a gel for the same genome.arrow_forwardEvaluation of PCR product electrophoresed on 0.8% agarose gel shows non-specific bands. The appropriate modification for the next PCR reaction is to: a) increase the concentration of Taq polymerase b) increase the number of PCR cycles c) decrease the template denaturation temperature d) reduce the concentration of primersarrow_forward
- There are three classes of restriction enzymes; Class I, Class II and Class II. Nevertheless, Class II is most popular in recombinant technology. Explain the reason behind this.arrow_forwardBelow is the result of PCR products running on agarose gel electrophoresis. You expect to see one band, but the results show two bands, as shown below. Why is there a lower band? What are they? How can we avoid such a band?arrow_forwardIn PCR amplification reaction, 5 mM EDTA was erroneously added to your final reaction, and you did not get any products. What is a possible explanation for this (based on the properties of EDTA)?arrow_forward
- The final amount of each primer required in a PCR reaction is 25 picomol. If the total volume of the PCR reaction is equal to 100 µl and the stock concentration of each primer is equal to 0.0025 mM. Calculate the volume of stock primer that needs to be added in order to ensure a primer amount of 25 picomol.arrow_forwardPlease DESCRIBE, in outline form, the method you will use to select for bacterial cells that have taken up the pL311 plasmid, and to screen those cells for the presence of plasmids that are likely to contain a cloned gene. Be sure to mention the specific media you will use. In addition, please explain the rationale behind this specific selection and screening procedure. (Remember that you have available the following types of media: (i) media containing neither kanamycin nor X-gal, (ii) media containing BOTH kanamycin and X-gal, (iii) media containing tetracycline, and (iv) media containing ampicillin.arrow_forwardYou are performing an experiment using CRISPR-cas9 to genetically modify the LacZ gene of a culture of E. coli. After you run the experiment, you decide to use gel electrophoresis to genotype the different bacterial cultures to determine if the gene editing was successful. How could your electrophoresis results confirm that the PCR was successful? And how could your electrophoresis results confirm that you successfully extracted genomic DNA from your bacterial samples?arrow_forward
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