Recombinant DNA Technology Quiz
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Questions and Answers

What is the primary function of recombinant DNA technology in research?

  • Studying the structure and function of genes in various organisms. (correct)
  • Improving the efficiency of food production.
  • Creating new types of microorganisms.
  • Developing new methods for bacterial cell growth.

What specific biological technique is used to visualize DNA molecules based on their size?

  • Microscopy.
  • Spectrophotometry.
  • Agarose gel electrophoresis. (correct)
  • Centrifugation.

What are the two main types of pipettes mentioned in the context of this practical?

  • Automatic and glass. (correct)
  • Automatic and manual.
  • Glass and plastic.
  • Micropipettes and macropipettes.

What is the primary benefit of separating plasmid DNA from bacterial cells?

<p>To study the structure and function of the plasmid DNA itself. (D)</p> Signup and view all the answers

What is the significance of the bacterium Escherichia coli (E. coli) in the development of recombinant DNA technology?

<p>It is a model organism commonly used for genetic manipulation. (B)</p> Signup and view all the answers

What are the two primary applications of recombinant DNA technology mentioned in the context?

<p>Gene identification and protein production. (B)</p> Signup and view all the answers

Which of the following is NOT a transferable research skill mentioned in the context?

<p>DNA sequencing. (A)</p> Signup and view all the answers

What is the primary purpose of the online practical described in the text?

<p>To teach students how to purify plasmid DNA from bacterial cells. (B)</p> Signup and view all the answers

What is the purpose of the glycerol in the loading buffer?

<p>To increase the density of the DNA sample, ensuring it sinks into the well. (D)</p> Signup and view all the answers

What is the purpose of the bromophenol blue in the loading buffer?

<p>To provide a visual marker for the progress of electrophoresis. (D)</p> Signup and view all the answers

What is the purpose of the DNA markers (M) in the gel?

<p>To serve as a reference for the size of the extracted plasmid DNA. (B)</p> Signup and view all the answers

Why is it important to remove all ethanol from the DNA sample before resuspending it in TE buffer?

<p>Ethanol can prevent the DNA from dissolving in the TE buffer, hindering further analysis. (A)</p> Signup and view all the answers

What is the main purpose of using an agarose gel in this experiment?

<p>To visualize and separate DNA fragments based on their size. (D)</p> Signup and view all the answers

What is the function of the TE buffer?

<p>To dissolve the DNA sample and provide a suitable environment for its storage. (B)</p> Signup and view all the answers

Why is a positive control (previously purified pBluescript DNA) used in this experiment?

<p>To validate the DNA extraction process and ensure that the extracted DNA is of sufficient quality. (D)</p> Signup and view all the answers

Why should gloves be worn when handling gels containing SafeView or SafeStain?

<p>To avoid skin irritation caused by the stain. (D)</p> Signup and view all the answers

What is the typical behavior of supercoiled plasmid DNA during gel electrophoresis?

<p>It runs faster than linear plasmid DNA. (B)</p> Signup and view all the answers

What is the result of RNA contamination in plasmid DNA preparations?

<p>RNA appears as a bright smear or cloud in the gel. (B)</p> Signup and view all the answers

In the presence of chromosomal DNA, where would this DNA appear on the gel compared to the 10 kbp marker fragment?

<p>It would run higher than the 10 kbp marker. (C)</p> Signup and view all the answers

Which form of plasmid is expected to be present in plasmid mini-preps?

<p>Both supercoiled and relaxed circular plasmid DNA. (A)</p> Signup and view all the answers

If chromosomal DNA is present during plasmid DNA preparation, what effect may this have?

<p>It may cause difficulty in distinguishing plasmid bands on the gel. (B)</p> Signup and view all the answers

What is the primary purpose of agarose gel electrophoresis in relation to DNA fragments?

<p>To estimate the size of DNA fragments (B)</p> Signup and view all the answers

Which of the following types of plasmid DNA migrates the fastest through an agarose gel?

<p>Supercoiled plasmid DNA (D)</p> Signup and view all the answers

Which statement describes relaxed or 'nicked' plasmid DNA?

<p>It has one strand nicked, resulting in a larger size for gel movement. (A)</p> Signup and view all the answers

What happens to plasmid DNA when both strands are cut at the same place?

<p>It converts to a linear form and has an intermediate migration rate. (A)</p> Signup and view all the answers

Which factor does NOT affect plasmid DNA migration through an agarose gel?

<p>Presence of RNA in the sample (B)</p> Signup and view all the answers

How does the molecular conformation of plasmid DNA influence its migration in gel electrophoresis?

<p>Different conformations lead to varying speeds in migration. (D)</p> Signup and view all the answers

What is the result of torsional stress being released in supercoiled plasmid DNA?

<p>It becomes relaxed and develops into a slower migrating form. (D)</p> Signup and view all the answers

In addition to plasmid DNA, which other types of genetic material might be observed on agarose gels?

<p>Contaminating chromosomal DNA and RNA (A)</p> Signup and view all the answers

What is the primary characteristic of plasmids?

<p>They replicate alongside the bacterial chromosome. (A)</p> Signup and view all the answers

Which strain of E.coli is typically used in laboratories for research and teaching?

<p>K12 strain (C)</p> Signup and view all the answers

What advantage do plasmids provide to bacterial cells?

<p>They allow for horizontal gene transmission. (B)</p> Signup and view all the answers

Which of the following best describes a plasmid with a high copy number?

<p>It can accumulate hundreds of copies in a single cell. (C)</p> Signup and view all the answers

What is the purpose of a plasmid mini-prep?

<p>To isolate plasmid DNA from bacterial cells. (B)</p> Signup and view all the answers

Which of the following is NOT a function of plasmids?

<p>Inducing mutations in the bacterial genome. (C)</p> Signup and view all the answers

What are pBluescript and pGLO used for in laboratory settings?

<p>As cloning vectors for DNA manipulation. (B)</p> Signup and view all the answers

Which statement correctly describes the size range of plasmids?

<p>Plasmids can range from approximately 1000 base-pairs to several thousand kilobase-pairs. (B)</p> Signup and view all the answers

What is the primary purpose of adding glucose in Solution 1?

<p>To increase osmotic pressure (A)</p> Signup and view all the answers

What role does EDTA play in Solution 1?

<p>Inhibits DNases by binding ions (B)</p> Signup and view all the answers

What occurs after adding Solution 2 to the pellet in the tube?

<p>Cells undergo lysis (B)</p> Signup and view all the answers

What component is present in Solution 2 that aids in the rupture of cells?

<p>Sodium hydroxide (B)</p> Signup and view all the answers

Why is it important to keep the tubes inverted for mixing after adding Solutions 1 and 2?

<p>To ensure even distribution of reagents (D)</p> Signup and view all the answers

What is the expected outcome after adding Solution 3 to the mix?

<p>Visible white precipitate formation (D)</p> Signup and view all the answers

Which of the following best describes the action of acetic acid in Solution 3?

<p>It neutralizes the pH to facilitate renaturation of DNA (D)</p> Signup and view all the answers

What characteristic is observed after the cellular debris precipitates in Solution 3?

<p>Presence of SDS and protein precipitate (B)</p> Signup and view all the answers

Flashcards

Plasmid DNA

A small, circular DNA molecule found in bacteria that can replicate independently of the main bacterial chromosome.

Plasmid DNA Purification

The process of extracting plasmid DNA from bacterial cells using various techniques, such as lysis and purification.

Agarose Gel Electrophoresis

A technique used to separate DNA molecules based on their size by applying an electric current through a gel matrix.

Bench-top Microcentrifugation

A technique that separates solid particles from a liquid by spinning the mixture at high speed.

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Molecular Cloning

The process of introducing foreign DNA into a host organism, typically bacteria.

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Gene Analysis

The study of the structure and function of genes in organisms.

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Production of Pharmaceuticals

The application of genetic engineering to produce pharmaceuticals, such as insulin and erythropoietin, using microorganisms or tissue culture cells.

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Escherichia coli (E. coli)

The bacterium commonly used in recombinant DNA technology and gene research.

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Supercoiled Plasmid DNA

The most compact form of plasmid DNA, with tightly coiled strands, leading to fast migration in a gel.

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Relaxed or 'Nicked' Plasmid DNA

The relaxed form of plasmid DNA, with a break or 'nick' in one strand, leading to slower migration in a gel.

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Linear Plasmid DNA

A form of plasmid DNA where both strands are cut at the same point, resulting in a linear shape with migration speed in-between supercoiled and relaxed forms.

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Estimating DNA Fragment Size

DNA fragments of different sizes move through the gel at different rates, allowing estimation of their lengths.

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Quantifying DNA Amounts

The amount of DNA in a sample can be determined by comparing the intensity of bands with known DNA concentrations.

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What are plasmids?

Extrachromosomal DNA molecules found naturally in many bacteria.

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What is plasmid DNA isolation?

The process of isolating plasmid DNA from bacteria, often referred to as a "mini-prep".

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What is agarose gel electrophoresis?

A technique that uses an electric current to separate DNA fragments based on their size, allowing visualization and analysis.

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What are cloning vectors?

Plasmids that have been engineered to carry and express foreign DNA, used for cloning and studying genes.

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What are genes encoded by plasmids?

Genes that provide bacterial cells with advantageous traits, such as resistance to antibiotics or the ability to break down heavy metals.

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How do plasmids move between bacteria?

Plasmids can be transferred between bacterial cells (horizontal transmission) or to daughter cells during cell division (vertical transmission).

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How do plasmids replicate?

Plasmids replicate differently, with some replicating once per cell division (low copy number) and others replicating autonomously (high copy number).

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Why are plasmids useful for molecular biologists?

Plasmids are valuable tools for molecular biologists, as they can be used for a wide range of applications, such as DNA cloning and protein expression studies.

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Solution 1

A solution containing glucose, Tris, and EDTA. Glucose increases osmotic pressure outside the cells, making them vulnerable to rupture. Tris buffers the pH, maintaining a consistent environment. EDTA binds ions crucial for DNA degradation enzymes, preventing DNA breakdown.

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Solution 2

This solution combines sodium hydroxide and a detergent (SDS). The alkaline mixture ruptures the cells, and the detergent disrupts the cell membrane, solubilizing proteins. NaOH denatures DNA into single strands.

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Solution 3

A solution containing acetic acid and potassium acetate. The acid neutralizes the pH, allowing DNA strands to renature. The salt precipitates SDS from solution, along with cellular debris, forming a visible precipitate.

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Cell Lysis

The process of breaking open cells to release their contents, including DNA.

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What are the different forms of plasmid DNA?

The different forms of plasmid DNA are supercoiled, relaxed and linear. Supercoiled is the most compact form, resulting in the fastest movement through the gel. Relaxed is nicked, meaning there is a break on one strand and is slower in the gel. Linear DNA is a form of DNA where both strands are cut. It moves at a speed in between the supercoiled and relaxed forms.

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What is RNase and why is it important in gel electrophoresis?

Ribonuclease or RNase is a protein that degrades RNA molecules. By degrading RNA, it will prevent RNA from contaminating and interfering with DNA in the gel. RNA is usually smaller than plasmid DNA, so if not degraded, it will run ahead of markers as a bright smear.

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What is the difference between linear and circular DNA?

Linear DNA has two free ends, whereas a circular plasmid has all its ends linked together.

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What causes a 'nick' in a plasmid?

A 'nick' in a circular plasmid occurs when one of the strands is broken, resulting in a relaxed circular form.

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Why would chromosomal DNA appear higher on the gel than plasmid DNA?

Chromosomal DNA is significantly larger than plasmid DNA. Therefore, if chromosomal DNA is present, it will run further than the 10kb marker on the gel. It may appear as a smear if it has been degraded during preparation.

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Ethanol removal during DNA extraction

Ethanol can interfere with DNA dissolving, so it's crucial to remove any residual ethanol by flicking the tube to ensure a successful DNA extraction.

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Purpose of TE buffer

TE buffer, containing Tris and EDTA, is used to resuspend DNA pellets after they've been dried. It helps dissolve the DNA and protects it from degradation.

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Trouble dissolving DNA pellets

Pure DNA dissolves easily in water, so if you encounter difficulties dissolving a pellet, it's likely that other cellular components are interfering with the process.

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Ideal Agarose Gel Concentration for Plasmid DNA

A 0.8% agarose gel concentration is suitable to see the plasmid DNA. This concentration allows for clear separation of the DNA bands based on size.

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Purpose of SafeView or SafeStain

SafeView or SafeStain is a fluorescent dye added to agarose gels to make DNA bands visible under a UV light. This helps identify the location of the separated DNA fragments.

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What are DNA Markers (M)?

Molecular weight markers, also known as DNA ladders, are mixtures of DNA fragments with known sizes. These markers are loaded into a gel to provide a reference for estimating the sizes of unknown DNA fragments.

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Purpose of Loading Buffer

Loading buffer, containing glycerol and a tracking dye, is added to the DNA sample before loading it into the gel. This prevents it from floating out of the wells and helps to track the progress of electrophoresis.

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Study Notes

Plasmid DNA Purification

  • Plasmid DNA is extra-chromosomal, circular DNA found in bacteria.
  • It's not essential for bacterial survival but often contains beneficial genes (e.g., antibiotic resistance).
  • Plasmids replicate independently of the bacterial chromosome (low or high copy number).
  • Plasmids are used in many laboratory procedures, like DNA cloning.

Online Practical 5

  • Students will isolate plasmid DNA from E. coli bacteria.
  • Methods to visualize plasmid DNA will be used.
  • This process includes separating DNA molecules based on size (agarose gel electrophoresis).
  • Essential microcentrifugation, pipetting techniques to isolate plasmid DNA are skills to be learned.

Agarose Gel Electrophoresis

  • Separates nucleic acids (DNA, RNA) based on size.
  • Agarose gel acts as a molecular sieve (matrix of holes).
  • Smaller fragments move faster than larger ones.
  • DNA is negatively charged, moving towards the positive electrode.
  • SafeView dye (binds to DNA/RNA) makes fragments visible under UV light.
  • DNA size estimation and quantification are possible.

Plasmid DNA Conformations

  • Plasmid DNA in bacteria is often supercoiled (coiled tightly).
  • Relaxed or 'nicked' form results from a strand break in the DNA molecule.
  • Linear DNA, cut, moves slower than supercoiled in the agarose gel.

Experimental Procedure

  • Overnight E. coli cultures are the starting material.
  • Cells are harvested, lysed (broken open).
  • Cellular debris is removed to isolate the plasmid DNA.
  • DNA is precipitated.
  • Ethanol washes are used to purify the DNA.
  • The clean plasmid DNA is resuspended in TE buffer.
  • Results (plasmid conformations, size estimations) are analyzed.

Gel Analysis

  • The results from the gel are visualized.
  • Chromosomal DNA, that has been degraded and is small, might also appear.
  • RNA is present in the original E. coli, running independently.
  • Markers (DNA fragments of known size) help interpret unknown sizes.

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BIOL10401 Online Practical 5: Plasmid DNA Purification PDF

Description

Test your knowledge on recombinant DNA technology and its applications in research. This quiz covers essential techniques, the significance of E. coli, and practical skills related to DNA visualization and pipetting. Perfect for biology students looking to deepen their understanding of genetic engineering.

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