18 Questions
What is a specialized version of the general technique of equilibrium or density gradient centrifugation?
Ethidium bromide-Cesium chloride density gradient
At what density does DNA band in a CsCl density gradient during centrifugation?
1.70 g/cm3
Which macromolecule floats at the top of the tube during density gradient centrifugation?
Protein
How can supercoiled DNA be separated from non-supercoiled molecules using density gradient centrifugation?
In the presence of ethidium bromide
If the alkaline denaturation method is employed, is further purification by organic extraction or column chromatography necessary?
No, it may not be needed
What is used to create a density gradient in density gradient centrifugation?
Cesium chloride
What is the primary mechanism by which ethidium bromide binds to DNA?
Intercalating between adjacent base pairs
How does the binding of ethidium bromide affect the buoyant density of supercoiled DNA compared to linear DNA?
Supercoiled DNA has a lower buoyant density due to the unwinding caused by EtBr binding
What is the purpose of using a CsCl gradient in the purification of plasmid DNA?
To separate plasmid DNA from linear bacterial DNA
What is the purpose of using ultraviolet radiation in the plasmid DNA purification process?
To induce the fluorescence of the EtBr bound to the DNA
What is the primary advantage of using the CsCl-ethidium bromide gradient centrifugation method for plasmid DNA purification?
It results in a highly pure plasmid DNA preparation, virtually 100% pure
What is the purpose of the dialysis step in the plasmid DNA purification process?
To remove the CsCl used in the density gradient centrifugation
Which of the following statements about the cell disruption process is true?
EDTA and lysozyme treatment is carried out in the presence of 25% sucrose to prevent immediate cell bursting
What is the primary reason for using a non-ionic detergent like Triton X-100 during cell lysis?
To cause minimal breakage of the bacterial chromosomal DNA
Why is centrifugation alone not sufficient for separating plasmid DNA from chromosomal DNA?
Both a and c
What is the primary advantage of using gentle cell lysis techniques?
It minimizes the breakage of chromosomal DNA
What is the purpose of using EDTA during the cell lysis process?
To chelate divalent cations and weaken the cell wall
Which of the following statements about the centrifugation step is correct?
It takes advantage of the physical attachment between bacterial chromosomes and cell envelopes
Explore the specialized technique of density gradient centrifugation using CsCl solution to separate macromolecules. Learn about the process of producing a density gradient through high-speed centrifugation.
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