RP Gen Lab

Report 1:
In the procedure section, we use an additional heating step to increase performance and
reduce waiting time. This is also one of the ways to soften. In addition, we use glycerin 10%
instead of water because glycerin acts as a clearing agent, it allows for the penetration of light
through the specimen making the cells and structures more visible. Glycerin also helps to
preserve the specimen by preventing dehydration and shrinking. A concern of 10% is typically
used as it strikes a balance between preservation and visualization.
Report 2:
In DNA extraction:
• Explain the function of the Sodium dodecyl sulfate (SDS) buffer?
For DNA extraction, because it could destroy non- covalent bonds of protein, which means
it can be used to destroy the cell membrane, denature the protein structure, and break the link
between proteins.
• Function of sodium acetate?

Sodium acetate is used to precipitate DNA. Naturally, DNA will easily dissolve in water,
to prevent that from happening, Sodium acetate with its positive charge Na , will bind to
+
negative PO of DNA and decrease its hydrophilicity.

3
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• What ís the components in the upper, inter and lower region?

+Lower: contain hydrophobic components such as lipids and phenol.
+Inter: contains denature protein.
+Upper: contains DNA and polar molecules.
• Does proteinase K destroy itself? Why?

Proteolytic enzymes, also known as proteinases or peptidases, are enzymes that
catalyze the breakdown of proteins into smaller peptides and amino acids. These
enzymes have a specific three-dimensional structure that allows them to selectively
bind to and hydrolyze specific peptide bonds in other proteins. The active site of the
enzyme, where the hydrolysis takes place, is specifically designed to recognize and
bind to the substrate (the protein being broken down) and not the enzyme itself. This
specificity allows the enzyme to degrade other proteins without degrading itself.
• Why do we mix isoamyl in the chloroform solution rather than using pure chloroform?
+By adding isoamyl alcohol, proteins are denatured and separated from the DNA, allowing
for better recovery of the nucleic acid.
+Isoamyl alcohol aids in separating the aqueous and organic phases during the extraction
process. It helps to form a distinct organic layer, which allows for easy separation of the
DNA-containing aqueous layer from other cellular components present in the sample.
+The addition of isoamyl alcohol can help reduce foaming during the extraction process
• What is the lysis buffer pH range for DNA extraction?

A commonly used pH range for lysis buffers is between pH 7.0 and pH 8.0.
In DNA qualification and quantification:
• What are the electrophoresis buffer functions?
+The electrophoresis buffer provides ions that enable the conduction of electric current
through the gel. This allows for the movement of charged DNA molecules within the gel
during electrophoresis.
+ The electrophoresis buffer helps maintain a stable pH environment during the
electrophoresis process.
+The presence of buffering agents in the electrophoresis buffer helps keep the DNA
fragments soluble and prevent their precipitation or denaturation during electrophoresis.
This is particularly important for maintaining the integrity of the DNA molecules and
ensuring accurate separation based on size.
• What are the standards to consider a quality DNA for qualification and quantification?
+Purity: A high-quality DNA sample should have minimal contamination from other
biological materials, such as proteins, RNA, or chemicals used during the extraction
process.
+Concentration: The concentration of DNA refers to the amount of DNA present in a given
volume. A high-quality DNA sample should have a sufficient concentration for the
intended application.
+Integrity: The integrity of DNA refers to the preservation of its structure without
degradation or fragmentation. High-quality DNA should exhibit minimal degradation,
especially for applications that require long DNA fragments or intact genetic material.
• What is the alternative method for electrophoresis?

+Spectrophotometry is an important technique used in many biochemical experiments that
involve DNA, RNA, and protein isolation, enzyme kinetics and biochemical analyses.
Since samples in these applications are not readily available in large quantities, they are
especially suited to being analyzed in this non-destructive technique. In addition, precious
samples can be saved by utilizing a micro-volume platform where as little as 1uL of sample
is required for complete analyses. A brief explanation of the procedure of
spectrophotometry includes comparing the absorbency of a blank sample that does not
contain a colored compound to a sample that contains a colored compound.
+In addition, microfluidic chip electrophoresis, also known as lab-on-a-chip
electrophoresis, utilizes a miniaturized platform where the electrophoresis process takes
place within microchannels. The chip contains integrated electrodes, sample loading wells,
and separation channels. DNA samples are loaded into the designated wells, and an electric
field is applied to drive the DNA fragments through the microchannels, separating them
based on size.
• What is the difference between Agarose and Polyacrylamide gel electrophoresis?
Gel Matrix Composition: Agarose gel electrophoresis uses agarose, a polysaccharide
derived from seaweed, as the gel matrix. Polyacrylamide gel electrophoresis uses
polyacrylamide, a synthetic polymer, as the gel matrix.
Pore Size and Resolution: Agarose gels typically have larger pore sizes, making them
suitable for separating larger DNA fragments, Polyacrylamide gels have smaller pore sizes
and can achieve higher resolution, enabling the separation of smaller DNA fragments.
Gel Preparation and Handling: Agarose gels are relatively easier to prepare and handle
compared to polyacrylamide gels. Agarose is typically mixed with a buffer and heated to
form a gel, which can be easily poured into gel trays. Polyacrylamide gels, on the other
hand, require monomer solutions and a polymerization process using a chemical initiator.
The preparation of polyacrylamide gels requires more precision and attention to detail, as
it involves the use of toxic acrylamide monomers and the need for adequate safety
measures.
• What is the relation between DNA length and electrophoresis?
The relation between DNA length and electrophoresis is based on the principle that DNA
molecules migrate through a gel matrix in an electric field during electrophoresis. The
movement of DNA fragments is influenced by their size and charge, which affects the rate
at which they migrate through the gel. As a result, during electrophoresis, DNA fragments
separate based on their size, with smaller fragments traveling faster and migrating farther
from the origin compared to larger fragments. This separation allows for the visualization
and analysis of DNA fragments of different sizes.
Report 3:
Why do we need to work with fruit flies?
Despite the significant evolutionary distance between fruit flies (Drosophila melanogaster)
and humans, there are several similarities between their genes. These similarities have been
instrumental in using fruit flies as a model organism to study various aspects of human biology:
-Fruit flies have a relatively simple and well-characterized genome. They have a short life
cycle, reproduce quickly, and produce a large number of offspring. These characteristics make
them highly used to genetic manipulation techniques, allowing researchers to introduce specific
mutations, knock out or overexpress genes, and study their effects.
-Despite the evolutionary distance between fruit flies and humans, many fundamental
biological processes and pathways are conserved between the two species. Genes involved in
processes such as cell division, tissue development, metabolism, and neurotransmission often have
functional counterparts in humans. By studying these processes in fruit flies, researchers can gain
insights into basic biological mechanisms that are relevant to human biology and disease.
-Fruit flies have homologs of numerous human disease-associated genes. By introducing
disease-causing mutations or manipulating these genes in fruit flies, researchers can create models
of human diseases.
-Using fruit flies as a model organism is advantageous from an ethical standpoint. Fruit
flies have a short lifespan and minimal cognitive complexity, minimizing potential ethical
concerns associated with animal research.

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Report 1:

• Function of sodium acetate?

negative PO of DNA and decrease its hydrophilicity.

• What ís the components in the upper, inter and lower region?

• Does proteinase K destroy itself? Why?

• What is the lysis buffer pH range for DNA extraction?

• What is the alternative method for electrophoresis?

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