5. (a) Without splitting a sample of peripheral blood mononuclear cells into two

Question

5. (a) Without splitting a sample of peripheral blood mononuclear cells into two different (5 marks) (b) Chloramphenicol had been used in plasmid amplification? What is its function tubes, how would you extract DNA and total RNA from the white blood cells? and why is it no longer required? (2 marks) (c) Briefly explain the major differences between pulsed field and standard agarose gel electrophoresis. (3 marks) 6. (a) In 1st strand cDNA synthesis during cDNA library construction, random primers are sometimes used. Why and what precautions must be considered? (4 marks) (b) Two different approaches could be used in 2nd strand cDNA synthesis. What are they and briefly discuss their pros and cons? (4 marks) (c) Blunt-ended DNA are required for ligation of adaptors. What enzymes (give 2 examples) could be used and how do they generate blunt ends? (2 marks)

Explanation / Answer

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Answer 5:

Part a) While WBCs are used to collect both DNA and RNA, both these samples can be collected without splitting the master sample into two parts. The WBCs can be lysated in cell lysis solution briefly to release all the cytoplasmic as well as nuclear contents in the medium. This medium now contains both DNA and RNA. This medium can be directly subjected to tri-reagent extraction by putting the sample on ice with tri-reagent followed by centrifugation. Formation of three layers would be observed. The top layer contains the RNA, the middle layer contains DNA and the bottom layer contains the whole cellular protein.

Part b) Chloremphenicol was previously used to enhance the extraction quality of plasmids which were present in low copy number in the host cell. This antibiotic chemically assisted the extraction and accumulation of these plasmids and hence used routinely. However, with the modernization of biotechnology and improvement in extraction methodologies, simple strategic extractions of the plasmids can be performed without using such antibiotics.

Part c) Pulse field electrophoresis is a technique used to resolve samples of very large molecular weight, such as DNA from whole organisms with millions of base pairs. This technique was evolved based upon the fact that the normal electric field and voltage used in the agarose gel electrophoresis is suitable only for resolving nucleic acids of low molecular weight. However, under such conditions, nucleic acids of large molecular weights remain intact and congested in the top layer of the well of the gel and hence do not resolve. In order to resolve this problem, a very continuous strong field of electric field is applied to the sample. This causes the nucleic acid to move continously in the gel and thud undergo resolution.

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