Centrifugation Techniques for Virus Isolation

Questions and Answers

What method can be used to isolate viral DNA effectively?

Chemical extraction followed by evaporation

Enrichment of virus particles through centrifugation prior to proteolytic digestion (correct)

Electrophoresis to separate based on charge

Filtration through a microfilter

Which type of centrifugation allows particles to reach their density isopycnic position?

High speed centrifugation

Isopycnic centrifugation (correct)

Rate zonal centrifugation

Sequential centrifugation

During the DNA extraction process, what role do proteases play?

They enhance silica binding

They cause DNA fragmentation

They release nucleic acids (correct)

They degrade nucleic acids

What is a key difference in RNA isolation compared to DNA isolation?

RNA isolation faces challenges due to the presence of RNases Signup and view all the answers

Which centrifugation technique separates particles based on their size and sedimentation coefficient?

Rate zonal centrifugation Signup and view all the answers

What is the main purpose of using affinity chromatography in nucleic acid extraction?

To selectively bind and purify nucleic acids Signup and view all the answers

What happens to nucleic acids under high salt conditions during extraction?

Nucleic acids bind tightly to the silica matrix Signup and view all the answers

Which of the following describes the composition of the solution before centrifugal separation?

Whole virus, macromolecules, and cell fragments of varying densities Signup and view all the answers

What is the primary purpose of using phenol/chloroform in nucleic acid extraction?

To remove proteins and hydrophobic components Signup and view all the answers

Which of the following factors is NOT essential for precipitating DNA with alcohol?

Type of alcohol used Signup and view all the answers

What effect does shearing have on high molecular weight DNA during extraction?

Creates smaller, less viscous fragments Signup and view all the answers

How can circular DNA plasmids be isolated effectively after alcohol precipitation?

Using isopycnic centrifugation in an EtBr-CsCl gradient Signup and view all the answers

What is the significance of EtBr intercalation in the context of DNA analysis?

It alters the buoyant density of DNA Signup and view all the answers

What is a recommended precaution to avoid shearing high molecular weight DNA?

Using a small diameter pipette Signup and view all the answers

Why is it important to purify plasmid DNA using a density gradient?

To separate different conformations of plasmid DNA Signup and view all the answers

What characteristic do fragments of DNA less than 10 kb exhibit regarding shearing forces?

They remain intact despite shearing Signup and view all the answers

Study Notes

Extraction of Nucleic Acids

Phenol/chloroform used to remove proteins and hydrophobic components from the aqueous phase.
Alcohol precipitation involves using 2.5 volumes of ethanol or 1 volume of isopropanol, along with high salt concentrations (e.g., 0.3 M sodium acetate) for DNA isolation.
Desalting process employs 70% ethanol to eliminate low molecular weight hydrophilic contaminants.

Chromosomal DNA Isolation

Chromosomal DNA can form large, viscous aggregates that are visible and can be spooled using a glass rod.
Typically consists of very long strands, can fragment into 50-100 kb sizes, and is prone to shearing.
To prevent shearing: Avoid small pipette tips and vigorous agitation.
Gene diagnostics tools (e.g., PCR) remain effective with high-molecular-weight DNA despite shearing,

Circular DNA/Plasmids

Plasmids exhibit diverse structures and conformations and are analyzed through DNA binding dyes like ethidium bromide (EtBr).
EtBr intercalates into double-stranded DNA, affecting buoyant density based on DNA conformation.
Plasmid purification can be achieved through isopycnic centrifugation in an EtBr-CsCl gradient, segregating particles based on density.

Cesium Chloride (CsCl) Density Gradient

CsCl creates a linear density gradient during ultracentrifugation, enhancing separation efficiency.
Macromolecules migrate to their equilibrium positions under sufficient ultracentrifugation forces without pre-forming the gradient.

Isolation of Viral DNA

Virus particles are concentrated via centrifugation before proteolytic digestion or chemical breakdown.
Employs multiple steps of sequential centrifugation at increasing g-forces to separate heterogeneous particles.
Isopycnic and rate zonal centrifugation methods allow for further separation based on density, size, and shape of viral particles.

DNA Extraction Kits

DNA extraction kits streamline nucleic acid isolation through standardized protocols.
Nucleic acids are released using proteolytic enzymes and purified via affinity chromatography columns that preferentially bind negatively charged nucleic acids depending on salt and alcohol concentrations.
RNA purification can occur simultaneously, and co-purified RNA can be subsequently digested with RNase.

RNA Isolation

RNA isolation follows similar protocols to DNA extraction but requires specific precautions due to the high prevalence of RNases in cells, which can degrade RNA.

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Description

This quiz covers the methods of centrifugation, particularly ultracentrifugation, for isolating and purifying viral DNA and nucleic acids. It focuses on the use of cesium chloride and the enrichment of virus particles through sequential centrifugation processes. Test your knowledge on these essential laboratory techniques!