Nt1310 Unit 1 Exercise 1
RANELLE JANINE L. ASI
BIO 120 S-5L
EXERCISE 9. Genomic DNA Isolation & Exercise 10. Polymerase Chain Reaction (PCR)
Cell and molecular biology is a science based on the various systems of a cell resulting to its regulation, maintenance and function. Many of these systems involve genetic information hence the study of the DNA is an essential part of this field. To able to analyze DNA, it must first be isolated and purified from its natural environment filled with biological molecules and compounds that cause physical or chemical interference in an experimental set-up. Several protocols have been established to efficiently extract DNA, one of which, the CTAB method, was performed and studied in this exercise. (De la Parte and Dita, 2014) As …show more content…
Denaturation is carried on by heating the double-stranded DNA at 94°C to separate the complementary strands that will serve as template in further cyclings. Pre-denaturation is sometimes done at the same temperature to ensure complete separation of strands. Annealing then occurs upon rapid cooling of the solution, allowing oligonucleotide primers to hybridize to the template. In this phase, however, the single strands of the template are too long and complex to be able to completely reanneal spontaneously. The gene fragment to be amplified will completely form double-stranded fragments upon further cycling of this step and the extension step. The extension step involves heating of the reannealed DNA to 72°C, the temperature at which the thermostable DNA polymerase in the mix will operate most efficiently in synthesizing new DNA strands.
Gel Electrophoresis exposes the molecular sizes of different DNA fragments as the lightest or shortest fragments travel fastest down the gel and the heaviest or largest fragments travel most slowly and are left near the top part of the gel. In this run, samples A-F show almost identical bands, indicating that all six samples are the same DNA. Two bands are found in each well which implies that each sample has two differently sized DNA fragments. The higher bands are most likely genomic DNA and the lower, larger bands are DNA in similarly sized fragments in …show more content…
However, the DNA from the third to the seventh lane travelled down the gel as they were smaller due to fragmentation during amplification. The bands on the five lanes also travelled different distances indicating that fragment lengths vary as the DNA polymerase replicated (and eventually fragmented) the DNA strands at different loci where the primer attached on each species’ genome. The site where the primer attached for Species A was around 700 kb from the tip of the segment, and hence resulted to a fragment that is 700kbp long. Species B’s fragment size was about 550 kbp, Species C and D’s are the same at around 300 kbp (hence, they have same gene loci), and Species E’s was about 250 kbp. Negative controls in gel electrophoresis serve the purpose of indicating the presence of contaminants in the reaction mix used in the PCR or in the gel itself. In this run, there is no indication of any contaminants in the samples. REFERENCES:
De la Parte, E. M. & Dita, M. (2014) Basic Aspects of Molecular Biology and DNA Extraction [Lecture slides]. Retrieved from http://www.fao.org/fileadmin/templates/agphome/documents/Pests_Pesticides/caribbeantr4/10MolecularBiology.pdf
Gaikwad, A. B. (n. d.) DNA extraction: Comparison of Methodologies. Retrieved from http://www.nbprgr.ernet.in
McPherson, M. J. & Moller, G. S. (2006) PCR (2nd ed.) New York, NY: Taylor
BIO 120 S-5L
EXERCISE 9. Genomic DNA Isolation & Exercise 10. Polymerase Chain Reaction (PCR)
Cell and molecular biology is a science based on the various systems of a cell resulting to its regulation, maintenance and function. Many of these systems involve genetic information hence the study of the DNA is an essential part of this field. To able to analyze DNA, it must first be isolated and purified from its natural environment filled with biological molecules and compounds that cause physical or chemical interference in an experimental set-up. Several protocols have been established to efficiently extract DNA, one of which, the CTAB method, was performed and studied in this exercise. (De la Parte and Dita, 2014) As …show more content…
Denaturation is carried on by heating the double-stranded DNA at 94°C to separate the complementary strands that will serve as template in further cyclings. Pre-denaturation is sometimes done at the same temperature to ensure complete separation of strands. Annealing then occurs upon rapid cooling of the solution, allowing oligonucleotide primers to hybridize to the template. In this phase, however, the single strands of the template are too long and complex to be able to completely reanneal spontaneously. The gene fragment to be amplified will completely form double-stranded fragments upon further cycling of this step and the extension step. The extension step involves heating of the reannealed DNA to 72°C, the temperature at which the thermostable DNA polymerase in the mix will operate most efficiently in synthesizing new DNA strands.
Gel Electrophoresis exposes the molecular sizes of different DNA fragments as the lightest or shortest fragments travel fastest down the gel and the heaviest or largest fragments travel most slowly and are left near the top part of the gel. In this run, samples A-F show almost identical bands, indicating that all six samples are the same DNA. Two bands are found in each well which implies that each sample has two differently sized DNA fragments. The higher bands are most likely genomic DNA and the lower, larger bands are DNA in similarly sized fragments in …show more content…
However, the DNA from the third to the seventh lane travelled down the gel as they were smaller due to fragmentation during amplification. The bands on the five lanes also travelled different distances indicating that fragment lengths vary as the DNA polymerase replicated (and eventually fragmented) the DNA strands at different loci where the primer attached on each species’ genome. The site where the primer attached for Species A was around 700 kb from the tip of the segment, and hence resulted to a fragment that is 700kbp long. Species B’s fragment size was about 550 kbp, Species C and D’s are the same at around 300 kbp (hence, they have same gene loci), and Species E’s was about 250 kbp. Negative controls in gel electrophoresis serve the purpose of indicating the presence of contaminants in the reaction mix used in the PCR or in the gel itself. In this run, there is no indication of any contaminants in the samples. REFERENCES:
De la Parte, E. M. & Dita, M. (2014) Basic Aspects of Molecular Biology and DNA Extraction [Lecture slides]. Retrieved from http://www.fao.org/fileadmin/templates/agphome/documents/Pests_Pesticides/caribbeantr4/10MolecularBiology.pdf
Gaikwad, A. B. (n. d.) DNA extraction: Comparison of Methodologies. Retrieved from http://www.nbprgr.ernet.in
McPherson, M. J. & Moller, G. S. (2006) PCR (2nd ed.) New York, NY: Taylor