Molecular Biology: PCR, Gel Electrophoresis, and Cell Culture

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Questions and Answers

What type of equipment is used for solubilizing agarose?

a small microwave

What safety feature is built into the transilluminator?

The UV-light turns off automatically when the access door of the photodocumentation system is opened

What is the purpose of the electro-porator in the laboratory?

Transformation of bacteria, yeast and cell lines

What is the benefit of the copper-enriched stainless steel interior of the CO2-incubator?

It inhibits possible bacterial contaminants Signup and view all the answers

What type of microscope is used to study protein-trafficking and cytotoxic effects of secondary compounds?

An epi-fluorescence microscope Signup and view all the answers

Why is the microscope placed on an antivibratoric table?

To minimize vibrations during photo capturing Signup and view all the answers

What is the purpose of the refrigerated centrifuge in the laboratory?

Separating or collecting cells, as well as preparing electrocompetent cells and doing Midi-preps Signup and view all the answers

What are two important considerations for the design of a laboratory?

Proper arrangement of light and air, as well as passage for crossing of air if there is less number of windows Signup and view all the answers

What is the primary function of PCR in molecular biology?

To amplify specific or desired sequence of DNA nucleotides Signup and view all the answers

What is the main purpose of gel electrophoresis in molecular biology?

To isolate DNA, RNA, and proteins according to their molecular weight Signup and view all the answers

What is the significance of cell culture in biological research?

It allows cells to be grown in a controlled artificial environment for various applications, including gene therapy, cancer research, and vaccine production Signup and view all the answers

What is the effect of ethanol on cell membranes?

It disrupts the physical structure of cell membranes Signup and view all the answers

What is the primary function of SDS in protein analysis?

It denatures secondary and tertiary structure of proteins by breaking their disulphide bond Signup and view all the answers

What is the purpose of PBS in biological research?

It helps to maintain a constant pH and provides a buffer solution that matches the osmolarity and ion concentrations of the human body Signup and view all the answers

What is the role of sucrose in the isolation of chloroplasts?

It plays a vital role in the isolation of chloroplasts and prevents osmotic shock Signup and view all the answers

What is a gene mutation, and how does it occur?

A gene mutation occurs when the nucleotide sequence of the DNA is altered and a new sequence is passed on to the offspring, either through substitution, insertion, or deletion of one or a few nucleotides. Signup and view all the answers

What is the primary function of sodium chloride in cell culture?

It is used for common cell culture procedures Signup and view all the answers

What is the significance of correcting gene mutations?

Correcting gene mutations is important to restore gene function and control diseases. Signup and view all the answers

What is the purpose of the experiment using E.Coli and agar plates?

The experiment is used to demonstrate the effect of a chemical mutagen on bacterial growth. Signup and view all the answers

What is the mechanism used by yeast like Saccharomyces cerevisiae to repair DNA dimers?

photorepair mechanism Signup and view all the answers

What is the purpose of exposing one plate to UV light in the experiment?

to induce mutation Signup and view all the answers

What is the role of the incubator in the experiment?

The incubator is used to maintain the appropriate temperature for the bacterial strain. Signup and view all the answers

What is the purpose of using a sterile toothpick in the experiment?

The sterile toothpick is used to streak the bacterial culture onto the agar plates. Signup and view all the answers

What is the importance of isolating high purity plasmid DNA in molecular biology?

it is essential for cloning, DNA sequencing, transfection, and gene therapy Signup and view all the answers

What is the limitation of the boiling lysis method for isolating plasmid DNA?

the amount of supercoiled plasmid obtained is often less, making it unsuitable for transfection experiments Signup and view all the answers

What is the difference between the two agar plates used in the experiment?

One agar plate has the chemical mutagen, while the other does not. Signup and view all the answers

What is the role of the autoclave in preparing nutrient agar plates?

sterilizing the plates Signup and view all the answers

What is observed after a few days of incubation?

The growth of the bacteria on both plates is observed. Signup and view all the answers

Why is it essential to use a chemical mutagen in the experiment?

The chemical mutagen induces mutations in the bacterial DNA, allowing for the study of gene mutations. Signup and view all the answers

What is the purpose of comparing the yeast colonies on both plates?

to observe the effects of UV radiation on yeast growth and morphology Signup and view all the answers

What is the starting material used in the isolation of plasmid DNA?

3 ml overnight grown culture of E. coli DH5α Signup and view all the answers

What is the significance of incubating the yeast plates at an appropriate temperature?

it allows for optimal yeast growth Signup and view all the answers

What is the primary purpose of adding mineral oil to the top of each tube in PCR?

To prevent evaporation if using a thermal cycler without a heated lid Signup and view all the answers

What is the typical number of cycles recommended for PCR amplification?

25-30 cycles Signup and view all the answers

What is the purpose of the extension step during PCR?

To allow the DNA polymerase to synthesize new DNA strands Signup and view all the answers

What type of electrophoresis is used to evaluate the amplified DNA in PCR?

Agarose gel electrophoresis Signup and view all the answers

What is the primary application of the Comet assay technique?

Assessing DNA damage in single cell suspensions, particularly in the field of toxicology Signup and view all the answers

What is the principle of the Comet assay test?

Visualizing and measuring DNA strand breaks in individual cells using gel electrophoresis and microscopy Signup and view all the answers

What is the purpose of the lysis solution in the Comet assay?

To release DNA fragments from the cells Signup and view all the answers

What is the role of the electric field in the Comet assay?

To attract negatively charged DNA fragments towards the anode Signup and view all the answers

Study Notes

Polymerase Chain Reaction (PCR)

PCR is used to amplify specific DNA sequences for various applications like DNA fingerprinting, mutation screening, and detection of pathogens.
It is performed using a thermocycler, which involves denaturation, annealing, and extension steps to amplify the DNA sequence.

Gel Electrophoresis

Gel electrophoresis is used to isolate and identify DNA, RNA, and proteins based on their molecular weight.
It involves comparing the resultant bands with standard or marker bands to identify the molecules.

Cell Culture

Cell culture is a technique used to grow cells in a controlled artificial environment (in vitro) for various applications like gene therapy, cancer research, virology, and vaccine production.
It involves harvesting cells from their natural environment (in vivo) and growing them in a controlled laboratory setting.

Reagents Used in Cell Biology Lab

Ethanol: disrupts cell membrane structure, used as a disinfectant and antiseptic.
Iodine: essential trace element, used as a starch indicator, chemical reagent, and biological stain.
SDS-Sodium Dodecyl Sulphate: denatures secondary and tertiary structure of proteins by breaking their disulphide bonds.
PBS-Phosphate Buffer Saline: a buffer solution that maintains a constant pH, used to maintain osmolarity and ion concentrations in biological research.
Sucrose: plays a vital role in isolating chloroplasts, prevents osmotic shock, and creates a density gradient.
NaCl-Sodium Chloride: used in common cell culture procedures.

Cell Culture Facilities

CO2 incubator: used to culture cell lines, has a copper-enriched stainless steel interior that inhibits bacterial contaminants.
Class II biological safety cabinet: used for manipulating cells.
Stereomicroscope: used for observing cells.
Epi-fluorescence microscope: used for studying protein trafficking and cytotoxic effects of secondary compounds.

Gene Mutation

A gene mutation occurs when the DNA sequence is altered, leading to a new sequence being passed on to the offspring.
It can result from substitution, insertion, or deletion of one or a few nucleotides.
Gene mutations can lead to serious diseases like Sickle cell anemia, thalassemia, cancer, and more.

Mutation Induction

Yeast like Saccharomyces cerevisiae has a photorepair mechanism to repair DNA dimers.
Blocking this repair mechanism using UV radiations can induce mutations.
Bacterial culture can also be used to induce mutations using chemical mutagens.

Isolation of Plasmid DNA

Boiling lysis method: used to isolate plasmid DNA from bacterial culture (E. coli DH5α).
The isolated plasmid is suitable for most cloning experiments.

PCR Amplification

Typical cycling parameters: 25-30 cycles of amplification with denaturation, annealing, and extension steps.
The amplified DNA can be evaluated using agarose gel electrophoresis and ethidium bromide staining.

Comet Assay Test

A technique used to assess DNA damage in single cell suspensions.
It is a sensitive and rapid method for measuring DNA damage and repair in single eukaryotic cells.
The principle of comet assay involves gel electrophoresis to visualize and measure DNA strand breaks in individual cells.

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Description

This quiz covers the basics of molecular biology, including PCR, gel electrophoresis, and cell culture techniques. Learn about the principles and applications of these essential tools in biotechnology.