Prescott's Microbiology
11th Edition
ISBN: 9781260211887
Author: WILLEY, Sandman, Wood
Publisher: McGraw Hill
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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 Microbiology
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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- 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_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_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
- Explain why a positive control and negative control are included in PCR experiments. Explain the three steps involved in each cycle of polymerase chain reaction.Why is loading dye added to the DNA sample for gel electrophoresis? Explain the function of the following components in a PCR reaction:− Primer, dNTP, MgCl, Taq polymerase, buffer.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_forwardA PCR reaction was performed to amplify the XULA3 gene, which is bp 882-5,364 on a plasmid that is 11,719 bp. After the PCR, the product was digested with XhoI. There are XhoI sites on the plasmid at bp 1,434, 4,655, and 7,368. Calculate the size(s) that would result when the product is digested with XhoI. Then enter the size of the largest fragment (in bp).arrow_forward
- If a uidA amplicon generateed by PCR is 200bp and the DNA fragments resulting from the restriction digest fall with 1000bp and 4000bp, which gel should be more concentrated? a) Higher concentration agarose b) Lower concetration agarose?arrow_forwardYou want to clone a specific PCR amplicon. You have determined that the amplicon you want to clone has enzyme restriction sites for HindIII and EcoRI. After investigation you have seen that the pUC18/19 plasmid also have these enzyme restriction sites in its multiple cloning site (MCS on map included). After enzyme digestion your amplicon is 854 bp long. What length will the recombinant plasmid be after you have inserted your amplicon? Show a calculation.arrow_forwardThe process of PCR essentially revolves around three phases. Briefly describe these phases and the events that occur in them. Take note the temperature on which these phases take place.arrow_forward
- The primers used in colony PCR will target EGFP genes. Write down your expected results for each case. You should. present the amplicon size in your answer. 1) ligation 2) self ligation of two vectors 3) self ligation of two inserts 4) ligation of a vector with multiple inserts. also how many inserts can be ligated into a vector 5) ligation of one insert into a vectorarrow_forwardAll of the following are performed during restriction fragment length polymorphism analysis. 1. splitting of double-stranded into single-stranded DNA 2. gel electrophoresis 3. autoradiography 4. immersion in radioactive probes 5. digestion of DNA with restriction endonucleases 6. use of a positive charge to transfer single-stranded DNA from a gel to a membrane. The correct sequence of these operations is whatarrow_forwardYou have set up a recombinant DNA experiment using the plasmid PBR322 as the vector (see plasmid below). You use the BamHI restriction site on the plasmid to insert the target DNA. The plasmid is then used to transform E.coli colls Is the following statement True or False? Growth of the transformed cells on agar containing both ampicillin and tetracycline will eliminate any cells that do not contain a plasmid. Clal Hindlll EcoRI Pvul BamHI Pstl amp tet PBR322 -Sall ori rop Pvull True Falsearrow_forward
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