ons separo er, a in elec Q: Describe cloning procedure. O: What are the common a
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ons separo er, a in elec Q: Describe cloning procedure. O: What are the common and useful features of plasmid? Q: Define competent cells. (Electro competent and chemically competent) Q: What Q: Transformation efficiency is measured by cfu/ug Q: Descried "Genetic Engineering", "Antibiotic selection" procedure. Q: Function of pGLO, Arabinose in transformation experiment. Q: what are the components of LB culture medium? Q: List major differences between DNA and RNA. Q: What do you understand by genetic code triplet bases? Where are they present? Q: What do understand by Replication, Transcription and Translation? Q: What are the major steps in DNA isolation protocol? What is the function of SDS, Chloroform and ethyl alcohol in DNA isolation protocol? Q: What are the absorption maxima of DNA, RNA and Protein? Q: What is the function of Cacl2 on E. coli cells? QWhy E. Coli is most suitable organism for transformation? Q: How do you conclude that transformation occurred?Explanation / Answer
Answer 1: Buffer is useful in maintaining the pH of the DNA solution close to neutral. It consists of Tris-base, acetic acid and EDTA. Moreover, buffers also inhibit the nuclease like DNA and RNA nuclease. Buffer ions also help for the conduction of electric current.
Agarose is the separation medium.
Under the electric current, Electrode is used for the movement of the DNA from negative to the positive electrode as DNA is negatively charged.
Answer 2: 1. Digestion of plasmid using specific restriction enzyme.
2. Ligation of a target gene sequence (desired sequence) with has the same restriction site that of the vector.
3. Transformed in E.coli. select the desirable colonies, subculture and maintain the culture.
Answer 3: Common and useful features of plasmid:
Replication: ability to replicate in host cells. When foreign DNA linked to a vector containing an origin of replication along with vector replication, the desirable DNA also starts replicating.
Multiple cloning sites that allow foreign DNA to be conveniently inserted into the vector.
Selectable marker to select the host cells containing the vector.
Answer 4: Electroporation: competency can also achieve through the use of electrical pulses called electroporation. It uses the short pulse of electric charge to facilitate DNA uptake. This technique induces small pores in the membrane and this pores called as electrophoresis which allows molecules, ions and water to pass from one side of the membrane to another one.
Chemical transformation: bacteria which are able to uptake DNA are called competent and made by chemical treatment. It changes to structure and permeability of the cell membrane so that DNA can enter the cell. It used calcium chloride and heat shock (42C) to promote the entry of the DNA into cells.
Answer 5: the process by which bacterial cells take up naked DNA molecules is called transformation.
Answer 6: Yes. transformation efficiency is measured in colony forming unit per ?g DNA used. transformation efficiency for small plasmid is 1×108cfu/?g.
Answer 7: Genetic engineering procedure:
1. DNA extraction
2. Gene Cloning
3. Gene design
4. Transformation
5. Backcross breeding
Antibiotic selection procedure:
1. Transformation into E.coli. bacteria containing plasmid is grown on antibiotic plates.
2. Only plasmid colonies with selected antibiotics will grow.
Answer 8: Bacteria containing plasmids are grown on agar medium which contains ampicillin and arabinose. Colonies with plasmids express ampicillin and beta-lactamase. In presence of arabinose, transcription starts.
Answer 9: the component of LB:
NACL= 10g/litre
YEAST EXTRACT= 5g/litre
PEPTONE= 10g/litre
Answer 10: DNA have C-H bond while RNA has C-OH bond. Moreover, DNA contains A, G, C and T and RNA contain A, G, C and U.
Answer 11: The genetic triplet code called a codon. There are 64 amino acids out of which 3 codes for stop codons.
Answer 12: replication: transmission of chromosomal DNA from generation to generation which is achieved by replicating DNA, providing two complimentary copies of parental DNA strand for faithful distribution into each daughter cell with the help of DNA polymerase.
Transcription: The process from DNA to RNA is called transcription using DNA dependent RNA polymerase. In which DNA strand act as the template for the synthesis of mRNA.
Translation: The process from RNA to protein is called translation using RNA dependent RNA polymerase.
Answer 13: DNA extraction steps:
• Cell wall lysis to release DNA into the solution.
• Purification of the DNA by precipitating polysaccharides and proteins.
• Precipitation of DNA and resuspension in a buffer.
SDS is used to lysis the cell well.
Chloroform is used to denature the proteins in the sample. After chloroform treatment, proteins remain in organic phase and DNA comes out in aqueous solution.
Along with phenol, Ethanol is also used for precipitation of DNA.
Answer 14: DNA and RNA absorb at 260nm whereas protein absorbs at 280nm.
Answer 15: calcium chloride causes DNA to precipitate onto the surface of the cells, which is responsible for the same kind of change in the cell well that improves DNA binding.
Answer 16: E.coli is most suitable host organism as it can be easily grown, have fast growth kinetics and genetically easily manipulated.
Answer 17: Bacteria containing plasmids are grown on agar medium which contains specific antibiotics. Colonies on the plates confirm the transformation.
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