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Food Biotechnology > DNA Cloning > Flashcards

DNA Cloning Flashcards

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1
Q

What are the function of the following tools in genetic engineering?

  • Restriction enzymes
  • DNA ligase
  • Plasmids
  • Bacteriophages
A
  • Restriction enzymes: To snip specific genes from DNA
  • DNA ligase: To repair broken DNA and paste new genes
  • Plasmids: Engineered to carry genes of interest.
  • Bacteriophages: to carry recombinant DNA.
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2
Q

What is the purpose of EDTA in the preparation of DNA?

A
  • Protects the DNA from DNAases.
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3
Q

What are the stages of genetic engineering?

A
  1. Identify the gene
  2. Cut the DNA sequences that contain the gene
  3. Use a vector to carry the gene into host cells
  4. Induce the cell to activate and express the gene
  5. Extract and purify the protein.
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4
Q

What is the function of sodium dodecyl sulfate?

A
  • Dissolves the lipid components and cellular proteins of the cell.
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5
Q

What are the reagents/chemicals needed for the precipitation step of DNA preparation? State their purposes.

A
  • Salt: Neutralizes the phosphate backbone of DNA

- Ethanol: Low dielectric constant facilitates the interaction of Na+ ions and PO3- ions.

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6
Q

Explain how silica column works to separate nucleic acids and protein contaminants?

A
  • (-) charged backbone of nucleic acids are attracted to (+) charged silica particles on the membrane.
  • Chaotrophic salt conditions allows nucleic acid to selectively bind to silica membrane by hydrogen bonding while other protein passes through
  • Salt acts as a cation bridge.
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7
Q

Briefly explain the mechanisms of electrophoresis

A
  • DNA is placed in a well on a charged field.
  • DNA backbone is (-) charged and migrate to the positive pole
  • Smaller DNA migrates faster and vice versa.
  • DNA separated based on size.
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8
Q

What is the UV absorption maximum of nucleic acids?

A

260 nm

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9
Q

What are the two measures that is used to assess purity of nucleic acid?

A
  • A260/A280 (1.8-2.0)

- A260/A230 (secondary) (2.0-2.2)

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10
Q

Describe the PCI method.

A

The PCI method is a method of purification that uses phenol, chloroform and isoamyl alcohol. The phenol denatures the protein, the chloroform removes phenol and denatured protein, and the isoamyl alcohol reduces foaming.

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11
Q

What is the function of sodium dodecyl sulfate?

A
  • Dissolves the lipid components and cellular proteins of the cell.
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12
Q

What are the 5 most common cloning vectors?

A
  • Plasmids
  • Bacteriophage
  • Cosmids
  • Bacterial Artificial Chromosomes
  • Yeast Artificial Chromosomes
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13
Q

List 4 techniques used for the disruption (lysis) of cells. Briefly describe them.

A
  • Sonication: Use sonicator that produces high freq sound waves.
  • Liquid Homogenization: Use Homogenizer
  • Mechanical: Use blender or homogenizer
  • Freeze: Use Dry ice or liquid nitrogen.
  • Manual Grinding: Use pestle and mortar.
  • Enzymatic: Uses lysozyme
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14
Q

What is the pH of PCI method? Why is this pH desired?

A

Basic pH (7-8). The DNA will only partition into the aqueous phase when the pH is basic.

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15
Q

What is the function of a cloning vector?

A

To introduce a recombinant DNA into a host cell for replication and analysis.

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16
Q

What are the 4 conformations that a plasmid can appear in?

A
  • Supercoiled DNA
  • Relaxed Circular DNA
  • Nicked open-circular DNA
  • Linearized DNA
17
Q

What are the three must haves for plasmids that is used for cloning? Define the functions of these sites.

A
  • Origin of replication: A segment recognized by cellular DNA-Replication enzymes
  • Selectable markers: Allow host to grow on selective media for screening purposes.
  • Cloning Site: Site for the insertion of foreign DNA that will not disrupt replication and essential markers.
18
Q

What are the procedures of an average cloning experiment?

A
  • Identify and isolate the gene of interest.
  • Ligation with cloning vector
  • Transformation: Introduce vector into host cell
  • Screening.
19
Q

What is the definition of competency in genetic engineering?

A

The ability of a cell to take up extracellular DNA from its environment.

20
Q

Briefly describe the 5 methods of introducing recombinant DNA into a cell.

A
  • Chemical transformation: Incubate ligated product with competent E. coli followed by heat shock treatment.
  • Electroporation: Incubate cell with plasmid DNA and subject it to a high-voltage pulse, making the cell membrane becomes permeable to large molecules.
  • Protoplast fusion: Protoplasts contact each other and fuses together spontaneously or via fusing agent.
  • Microinjection: Injecting foreign DNA into cell using micropipette
  • Gene gun: Inject cells with genetic information.
21
Q

Describe the alkaline lysis method.

A
  1. Bacteria containing the desired plasmid are harvested from culture by centrifugation.
  2. The bacterial suspension is subjected to lysis by an alkaline solution containing SDS and NaOH.
  3. SDS lyse cells and denature proteins while the alkaline condition denatures genomic DNA, plasmid DNA and proteins.
  4. The mixture is neutralized by potassium acetate, plasmid DNA reanneals while genomic DNA form precipitate.
  5. Precipitate is separated by high speed centrifugation, plasmid from the supernatant is recovered by precipitation using isopropanol or ethanol.
22
Q

State 3 methods for the purification of plasmid DNA.

A
  • Phenol-Chloroform method
  • Spin column method
  • Ethanol precipitation
23
Q

Explain in detail, the chemical transformation method.

A
  • Cells are prepared in a solution of CaCl2 on ice.
  • Competent cells are incubated with recombinant DNA on ice.
  • Temperature is raised for a short time (heat-shock), recombinant DNA moves into the cell membrane.
  • Transformed cells are incubated to recover and grow.
  • Screening via antibiotic agar plates.
24
Q

What are the two important vectors discussed in this syllabus?

A
  • pBR322: Contains two marker genes conferring ampicillin and tetracycline resistance on E. coli
  • pUC19: Contains ampicillin resistance and a LacZ gene (galactosidase enzyme converts X-gal into blue-colored compound) - Blue white screening.

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