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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Textbook Question
Chapter 9, Problem 3A
The following picture shows bacterial colonies growing on X-gal plus ampicillin in a blue-white screening test. Which colonies have the recombinant plasmid? The small satellite colonies do not have the plasmid. Why did they start growing on the medium 48 hours after the larger colonies?
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Examine the pGLO plasmid DNA
solution with the UV lamp. Note your
observations. Using a micropipettor,
withdraw 10 ul of plasmid and mix it
into the cell suspension of the
+PGLO tube by pipetting up and
down three times. Close the tube and
return it to the rack on ice. Also close
the -PGLO tube. Do not add plasmid
DNA to the -pGLO tube. Why not?
Since higher concentration colcemid will result in shorter chromosome, you want to
change your protocol and reduce final concentration of colcemid in your 10 ml blood
culture from 0.1ug/ml to 0.05ug/ml. how many ul colcemid stock solution with
concentration10ug/ml needed to be added in 10ml blood culture?
Dr. Wakefield would like to isolate recombinant plasmids from her bacterial culture using the
alkaline lysis method. She is planning to use the chemicals as listed below:
Solution I: 50 mM glucose, 25 mM Tris-Cl (pH 8.0), 10 mM EDTA (pH 8.0)
Solution II: ???
Solution III: 5 M potassium acetate, glacial acetic acid, de-ion water
Ethanol 70% (v/v)
Isopropanol
TE-RNAase pH 8.0
(i)
(ii)
(iii)
Based on the chemical list above, state the content(s) of Solution II.
Explain the functions of Solution II described in Q3 a) (i) in plasmid isolation.
What is the role of alcohol precipitation conducted after the plasmids are obtained at
the end of the procedure? Discuss the roles of ethanol and salt in alcohol precipitation
step.
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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Need a deep-dive on the concept behind this application? Look no further. Learn more about this topic, biology and related others by exploring similar questions and additional content below.Similar questions
- A plaque assay is performed beginning with 1 mL of a solution containing bacteriophages. This solution is serially diluted three times by combining 0.1 mL of each sequential dilution with 9.9 mL of liquid medium. Then 0.1 mL of the final dilution is plated in the plaque assay and yields 17 plaques. What is the initial density of bacteriophages in the original 1 mL?arrow_forwardYou have just carried out a transformation using a plasmid (possessing a Amp-resistance gene) that was 0.001 ug/ul in concentration. You added 10 ul of this plasmid to 100 ul of a bacterial cell suspension. Then you added 250 ul of LB following heat shock. After plating 200 ul of this cell suspension with LB onto a LBA plate, you observe 8 colonies on this plate. What is the transformation efficiency of this plasmid (in colonies/ug of plasmid) based on these results? about 1400 O about 80O about 4000 O about 400O O about 8000 f6 米 IO fs fg f1o 19 08. O OOarrow_forwardYou have isolated genomic DNA and plasmid DNA from a bacterial culture. Please answer the following questions: 1. What solution did you use to elute DNA from the silica resin column? 2. What is the function of the chaotropic salt in DNA isolation? 3. Why do we add the chaotropic salt for genomic DNA before lysis and for plasmid isolation we add the salts after the lysis step?arrow_forward
- In the "Bacterial Transformation" experiment, why were there no fluorescence bacteria present in the other plates even though these were inserted by the +pGLO plasmid? (Note: Discuss the importance on the addition of ampicillin and arabinose to the medium of the genetically engineered bacteria). Limit your answer to 1-3 sentences only.arrow_forwardPlease answer the following using the experiment data below. Provide the formulas or methods used for calculations of each. Number of colonies on LB/amp/ara plate = Micrograms of DNA spread on the plates = Transformation efficiency = DNA plasmid concentration: 0.08 μg/μl 250 μl CaCl transformation solution 10 μl pGLO plasmid solution 250 μl LB broth 100 μl cells spread on agar 227 colonies of transformantsarrow_forwardSolution B that was used during the plasmid isolation contains 0.2 M NaOH (see practical manual). Which effect does NaOH have on E. coli DNA A) It denatures genomic DNA and plasmid DNA. B) It denatures genomic DNA and but not plasmid DNA. C) It denatures plasmid DNA but not genomic DNA. D) It denatures neither genomic DNA nor plasmid DNA. E) This is unpredictable.arrow_forward
- Solution B that was used during the plasmid isolation contains 0.2 M NaOH (see practical manual). Which effect does NaOH have on E. coli DNA A) It denatures genomic DNA and plasmid DNA. B) It denatures genomic DNA and but not plasmid DNA. C) It denatures plasmid DNA but not genomic DNA. D) It denatures neither genomic DNA nor plasmid DNA. E) This is unpredictable. Which statement on the migration of DNA fragments through agarose gels is false A) Small fragments migrate faster than larger fragments because they can move faster through the agarose pores. B) Large fragments migrate faster than small fragments because they carry more negative charges. C) DNA fragments migrate towards the positive pole. D) Supercoiled DNA may migrate significantly different through the gel than linear DNA of equal size. E) The higher the agarose concentration the better the separation of smaller fragments as compared to larger fragments.arrow_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_forwardAt what stage of the culture should bacterial colonies be harvested for plasmid DNA extraction? How about for genomic DNA extraction? What distinguishes the xanthogenate-based methodology from the traditional phenol/chloroform method for isolating DNA from bacteria? What makes potassium ethyl xanthogenate efficient in isolating DNA from a variety of microorganisms?arrow_forward
- Why are the following reagents used? Neutralizing solution (Plasmid isolation) Isopropanol (Plasmid isolation) RNase (isolation of genomic DNA)arrow_forwardA cloning vector map is shown below. EcoRI Bam Ban Hind P-galactosidase Amp Bam Bam EcoRI Ori C Which restriction site is best for inserting a DNA fragment for selection of chimeric plasmid containing colonies? 1) They're all equally good. 2) Hindll 3) EcoRI 4) BamHIarrow_forwardDefine the following terms: Plasmid Restriction Enzyme Standard Curvearrow_forward
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