Microbiology: An Introduction (13th Edition)
13th Edition
ISBN: 9780134605180
Author: Gerard J. Tortora, Berdell R. Funke, Christine L. Case, Derek Weber, Warner Bair
Publisher: PEARSON
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Chapter 9, Problem 1CAE
Summary Introduction
Introduction:
Polymerase chain reaction (PCR) is an in vitro amplification technique implemented to generate many copies of a given DNA fragment.
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biotechnology lab class :1. The bacterial streaking on antibiotic containing agar plates has been completed and the plates will be available for you to inspect in the lab class.2. You will complete a restriction enzyme digest on the four available plasmid stocks and then examine the products by agarose gel electrophoresis.
Photographs of the gel are attached to identify the plasmid present in each analysed sample.Now consider the following questions :- For each of the four plasmids used in the practical, identify the size (in kb) of the linear DNA fragment(s) that you would expect to obtain if the EcoRI digest is complete.- Using the provided negative image of the gel, which shows the bands present in the ND and D samples for each plasmid, identify the plasmid that was present in each of the four plasmid stock.- Explain how you were able to identify plasmid in each sample using the gel, considering how linear DNA fragments of different length are separated by agarose gel…
Part A. If student counts 63 colonies on their 10^-6 dilution LB plate. What was the original concentration of their cells if they plated 100ul?
Part B.If we used pGFPuv as the template for PCR positive control. This is because:
a. it contains the GFP gene so it should show a product.
b. It contains DNA fragments that were added to the ligation reaction.
c. It is the desired plasmid we wanted to make.
d. So we have a band to compare our unknown plasid to allowing us to check if the unknown is the right size.
2. Restriction Enzyme Mapping - use any resources to assist you including
the hints below.
A circular plasmid molecule (12,000 total base pairs in size) was cut with a
series of restriction enzymes and the digestions were size fractionated by
agarose gel electrophoresis. Some digestions involved just one enzyme (single
digest), some combinations of two enzymes (double digests), and one utilized all
three enzymes (triple digest).
Agarose gel electrophoresis of the digestions produced bands of the following
sizes.
Enzyme
EcoRI
Hindi!!
Pstl
EcoRI and Hindill
EcoRI and Psti
Hindill and Pstl
EcoRI, Hindill, and Pst I
Bands of the Agarose Gel (size in base pairs)
2300 and 9700
4000 and 8000
12,000
800, 1500, 2500, and 7200
2300, 3200, and 6500
4000
800, 1500, 2500, 3200, and 4000
I
Draw a plasmid map showing the location of the restriction enzyme sites
relative to each other for each map. Include all 7 maps in your answer.
Chapter 9 Solutions
Microbiology: An Introduction (13th Edition)
Ch. 9 - Compare and contrast the following terms: a. cDNA...Ch. 9 - Differentiate the following terms. Which one is...Ch. 9 - Some commonly used restriction enzymes are listed...Ch. 9 - Suppose you want multiple copies of a gene you...Ch. 9 - Which enzyme makes the smallest fragment...Ch. 9 - Describe a recombinant DNA experiment in two or...Ch. 9 - List at least two examples of the use of rDNA in...Ch. 9 - You are attempting to insert a gene for saltwater...Ch. 9 - How does RNAi silence a gene?Ch. 9 - Prob. 10R
Ch. 9 - Restriction enzymes were first discovered with the...Ch. 9 - The DNA probe, 3-GGCTTA, will hybridize with which...Ch. 9 - Which of the following is the fourth basic step to...Ch. 9 - The following enzymes are used to make cDNA. What...Ch. 9 - If you put a gene in a virus, the next step in...Ch. 9 - You have a small gene that you want replicated by...Ch. 9 - Pieces of human DNA stored in yeast cells. a....Ch. 9 - A population of cells carrying a desired plasmid....Ch. 9 - Self-replicating DNA for transmitting a gene from...Ch. 9 - A gene that hybridizes with mRNA. a. antisense b....Ch. 9 - Design an experiment using vaccinia virus to make...Ch. 9 - Why did the use of DNA polymerase from the...Ch. 9 - The following picture shows bacterial colonies...Ch. 9 - Prob. 1CAECh. 9 - Using the restriction enzyme ECORI, the following...
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- Part 4. Putting It Together 1) Consider the diagram below as well as the given information. This diagram represents a piece of circular DNA which was cut in 4 separate reactions (4 different test tubes, each with some of this DNA in it). One digest was done with AvaI, another with ClaI, a third with EcoRV, and a fourth with ScaI. The locations of the recognition sequences for each restriction enzyme are shown along with the location of that site in bp along the circle (it goes clockwise from position 1). You run an agarose gel with a molecular weight marker in the first lane, the AvaI digest in lane 2, the ClaI digest in lane 3, the EcoRV digest in lane 4, and the ScaI digest in lane 5. a) Use the space below and draw out the agarose gel described above. Use your drawing to answer the next questions. b) How many bands of DNA are there in lane 3? c) How many bands of DNA are there in lane 5? d) There would be 2 bands of DNA in lane 4. How big are they? e) Which lane…arrow_forwardprofile-image If you take a DNA photolyase- strain of E. coli, and subject it to the treatment below step 1, spread evenly on LB plate step 2, subject to UV light step 3, cover half of plate and grow in light What will be the most likely outcome if the level of UV is nearly lethal to cells on the plate? a. Only colonies on uncovered side of plate b. Only colonies on covered side of plate c. very few colonies in roughly equal numbers on both sides of platearrow_forward4. Animation 12.4: This table shows results from a genetic test conducted on DNA samples from two patients. The patients are a newly married couple seeking genetic counseling about their risks of passing on a genetic disease to any children they may have in the future. The samples included the DNA region including and immediately surrounding a single gene. Restriction enzyme cleavage analyses were performed on the samples. Female Patient 1) 3400 bp fragment Male Patient 1) 1600 bp fragment 2) 1800 bp fragment 3) 3400 bp fragment DNA fragments on gel What do the genetic test results indicate about this case? O Both the woman and the man are homozygous for the mutation. O Both the woman and the man are heterozygous. O The woman is homozygous for the mutation and the man is homozygous for the normal gene. O The woman is homozygous for the mutation and the man is heterozygous.arrow_forward
- A. DNA Sequencing 1. Determine the base sequences of the sample DNA from unknown organisms by examining the bands after gel electrophoresis on photographic films. a. Unknown 1 b. Unknown 2 AGT C I I c. Unknown 3 ||| || | | AGT | || | ||| | | | || I || || ull с I AGT 2. Write down the DNA sequences of each Unknown. I I || d. Unknown 4 || |||||| AGT || ||| | || I || ||| с C ||arrow_forwardDrag and drop the appropriate text in the gaps below. Firstly, the plasmid DNA in the E. coli is propagated through Then the bacterial cells are pelleted by centrifugation at 10,000 RPM. The media supernatant is removed and the bacterial cells are The bacterial cells are then lysed via |. Contaminating macromolecules such as protein and chromosomal DNA is removed by Overnight incubation of the bacterial culture washed in cell resuspension buffer addition of lysis buffer addition of neutralisation buffer washing of the spin column separation via agarose gel electrophoresisarrow_forwardDNA Cheek Cells Lab Identify the component of the solution which is the extracted DNAarrow_forward
- Kpnl 4. Plasmid Z has a size of 7 kb, and the map shows Kpnl (K) and Pstl (P) cut sites relative to each other. This plasmid was digested with three different restriction enzymes 2000 bp 3500 bp Kpnl (K), Pstl (P) and Bgll (B) either alone or in combination and the samples run on an agarose gel as shown below. Where does Bgll (B) cut this plasmid ? Does the plasmid have one recognition site or two for Bg|l? Describe the Bgll cut site in this plasmid relative to the Kpnl cut Plasmid Z -7 kb Pstl site. How many bases to the left or right of the Kpnl cut site would you observe the Bgll cut site. Explain briefly. 1500 bp Pstl Ladder Kpni Psti K/P Bgl K/B KPB 7000 bp 7000 bp 5600 bp 5500 bp 4900 bp 3500 bp 2000 bp 1500 bp 1500 bp 1500 bp 1400 bp 600 bp %3Darrow_forwardSelect all the following statements that are correct regarding agarose gel electrophoresis and restriction digests. Group of answer choices A higher percentage of agarose is used to distinguish between smaller pieces of DNA DNA fragments run from the positive electrode (where they are loaded) to the negative electrode Buffer is poured into the electrophoresis chamber AFTER the DNA has been loaded into the lanes. Larger DNA fragments migrate more slowly than smaller DNA fragments Loading dye is added AFTER restriction digests have been completed.arrow_forwardHersheyChase Experiments The graph shown in FIGURE 8.5 is reproduced from an original 1952 publication by Hershey and Chase. Bacteriophage were labeled with radioactive tracers and allowed 10 infect bacteria. The virusbacteria mixtures were then whirled in a blender to dislodge any viral components attached to the exterior of the bacteria. Afterward, radioactivity from the tracers was measured. FIGURE 8.5 Detail of Alfred Hershey and Martha Chases 1952 publication describing their experiments with bacteriophage. Infected bacteria refers to the percentage of bacteria that survived the blender. How did the researchers know that the radioisotopes in the fluid came from outside of the bacterial cells and not from bacteria that had been broken apart by whirling in the blender?arrow_forward
- HersheyChase Experiments The graph shown in FIGURE 8.5 is reproduced from an original 1952 publication by Hershey and Chase. Bacteriophage were labeled with radioactive tracers and allowed 10 infect bacteria. The virusbacteria mixtures were then whirled in a blender to dislodge any viral components attached to the exterior of the bacteria. Afterward, radioactivity from the tracers was measured. FIGURE 8.5 Detail of Alfred Hershey and Martha Chases 1952 publication describing their experiments with bacteriophage. Infected bacteria refers to the percentage of bacteria that survived the blender. After 4 minutes in the blender, what percentage of each isotope was extracellular?arrow_forwardHersheyChase Experiments The graph shown in FIGURE 8.5 is reproduced from an original 1952 publication by Hershey and Chase. Bacteriophage were labeled with radioactive tracers and allowed 10 infect bacteria. The virusbacteria mixtures were then whirled in a blender to dislodge any viral components attached to the exterior of the bacteria. Afterward, radioactivity from the tracers was measured. FIGURE 8.5 Detail of Alfred Hershey and Martha Chases 1952 publication describing their experiments with bacteriophage. Infected bacteria refers to the percentage of bacteria that survived the blender. The extracellular concentration of which isotope increased the most with blending?arrow_forwardHersheyChase Experiments The graph shown in FIGURE 8.5 is reproduced from an original 1952 publication by Hershey and Chase. Bacteriophage were labeled with radioactive tracers and allowed 10 infect bacteria. The virusbacteria mixtures were then whirled in a blender to dislodge any viral components attached to the exterior of the bacteria. Afterward, radioactivity from the tracers was measured. FIGURE 8.5 Detail of Alfred Hershey and Martha Chases 1952 publication describing their experiments with bacteriophage. Infected bacteria refers to the percentage of bacteria that survived the blender. Before blending what percentage of each isotope. 35S and 32P, was extracellular (outside the bacteria)?arrow_forward
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