Southern Blotting - Q&A Set
Q1. What does this setup demonstrate?
This setup shows the Southern blotting method, which finds a particular DNA sequence in a mixed DNA
sample. It consists of hybridizing with a tagged probe after transferring DNA fragments from a gel to a
membrane.
Q. 2 Who discovered this method?
Edwin Southern created the Southern blotting method in 1975. It is named for him.
Q. 3 What is the principle of this technique?
The hybridization concept underlies southern blotting. It consists of identifying particular sequences using
a tagged complementary DNA probe, transferring DNA fragments to a membrane, and gel electrophoresis
separating them.
Q. 4 What are the applications of this technique?
This method has several uses.
Finding gene presence or absence
Forensic science's DNA fingerprinting
Diagnosis of genetic diseases
Studies on gene mapping and genome organization
Confirmation of transgenic species
Q. 5 What are the main steps of this method?
The main steps of Southern blotting are
Restriction digestion and DNA extraction
Gel electrophoresis for DNA fragment separation
Melting of DNA into single strands
DNA movement to a nylon or nitrocellulose membrane
Labeled probe hybridization
Autoradiography or chemiluminescence-based detection
Q. 6 What type of probe is used in this technique?
Used is a single-stranded DNA probe complementary to the target sequence. Detection usually involves
labelling with non-radioactive markers like digoxigenin or biotin or radioactive isotopes.
Q7. Before transfer, why is DNA denatured?
DNA is denatured to split the strands so that the single-stranded probe may precisely hybridize to its
complementary sequence on the membrane.
Q. 8 How is the DNA visualized after hybridization?
The particular DNA sequence is bound by the tagged probe. The label kind determines visualization; X-
ray film (autoradiography) detects radioactive probes; non-radioactive probes are visualized using
chemiluminescence or colorimetric detection.
Q. 9 What are the limitations of Southern blotting?
Labor-intensive and time-consuming
Calls for significant quantities of high-quality DNA.
Uses radioactive materials (if relevant)
Not as sensitive as PCR-based techniques
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Q10. In what ways does Southern blotting differ from Northern and Western blotting?
Southern blotting: finds DNA
Northern blotting: finds RNA
Western blotting: identifies proteins
Northern Blotting – Q&A Set
Q1. What does this setup demonstrate?
This setup demonstrates the Northern blotting technique, which is used to detect and analyze specific
RNA molecules in a sample. It helps in studying gene expression at the mRNA level.
Q2. Who discovered this technique?
Northern blotting was developed by James Alwine, David Kemp, and George Stark in 1977, inspired by
the Southern blotting method for DNA.
Q3. What is the principle of this technique?
Northern blotting is based on nucleic acid hybridization. RNA is separated by gel electrophoresis,
transferred to a membrane, and hybridized with a labeled probe that is complementary to the target RNA
sequence.
Q4. What are the applications of this technique?
Studying gene expression levels across tissues or treatments
Detecting mRNA size and abundance
Investigating alternative splicing and RNA isoforms
Diagnosing viral infections or genetic diseases
Validating transcriptional responses in experiments
Q5. What are the main steps of Northern blotting?
RNA extraction from cells or tissues
Denaturing agarose gel electrophoresis to separate RNA by size
Transfer of RNA to a nylon or nitrocellulose membrane
Cross-linking or baking to fix RNA on the membrane
Hybridization with a labeled DNA or RNA probe
Detection using autoradiography or chemiluminescence
Q6. Why is a denaturing gel used in this technique?
A denaturing gel (typically containing formaldehyde) is used to prevent secondary structure formation in
RNA, ensuring that RNA molecules migrate according to size.
Q7. What kind of probes are used in Northern blotting?
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Labeled DNA or RNA probes that are complementary to the target RNA are used. These can be
radioactive (e.g., ^32P-labeled) or non-radioactive (e.g., digoxigenin-labeled).
Q8. How is RNA visualized in this technique?
After hybridization, the probe bound to the target RNA is detected through autoradiography (for
radioactive probes) or chemiluminescence/colorimetric detection (for non-radioactive probes).
Q9. What are the limitations of Northern blotting?
Requires high-quality, intact RNA
Less sensitive than RT-PCR or qPCR
Labor-intensive and time-consuming
Use of radioactive materials (in some versions)
Western Blotting – Q&A Set
Q1. What does this setup demonstrate?
This setup demonstrates the Western blotting technique, which is used to detect and analyze specific
proteins from a complex mixture, such as a cell lysate or tissue extract.
Q2. Who discovered this technique?
Western blotting was first described by W. Neal Burnette in 1981, adapting principles from Southern and
Northern blotting. The name “Western” was a playful nod to these earlier methods.
Q3. What is the principle of this technique?
Western blotting is based on the specific binding between an antibody and its target protein. Proteins are
separated by SDS-PAGE, transferred to a membrane, and then probed with primary and secondary
antibodies for detection.
Q4. What are the applications of this technique?
Detection and quantification of specific proteins
Verification of protein expression in cells or tissues
Diagnosis of diseases (e.g., HIV, Lyme disease)
Analysis of post-translational modifications
Validation of antibodies or gene knockdowns
Q5. What are the main steps of Western blotting?
Protein extraction from cells or tissues
SDS-PAGE (gel electrophoresis) to separate proteins by size
Transfer of proteins to a membrane (PVDF or nitrocellulose)
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Blocking to prevent non-specific binding
Incubation with primary antibody (specific to the target protein)
Incubation with secondary antibody (linked to a detection system)
Detection via chemiluminescence, fluorescence, or colorimetric methods
Q6. What is the role of SDS-PAGE in Western blotting?
SDS-PAGE separates proteins based on molecular weight. SDS denatures the proteins and gives them a
uniform negative charge, allowing size-based separation in the gel.
Q7. Why is blocking important in this technique?
Blocking prevents non-specific binding of antibodies to the membrane, reducing background noise and
ensuring accurate detection of the target protein.
Q8. What is the difference between primary and secondary antibodies?
Primary antibody: Binds directly to the target protein
Secondary antibody: Binds to the primary antibody and is linked to a detection system (e.g., HRP for
chemiluminescence)
Q9. How is the signal detected?
Detection depends on the label on the secondary antibody. Common methods include:
Chemiluminescence (e.g., HRP + ECL substrate)
Colorimetric detection (e.g., DAB or BCIP/NBT)
Fluorescence using fluorescently-labeled antibodies
Q10. What are the limitations of Western blotting?
Time-consuming and multi-step
Requires high-quality, specific antibodies
Semi-quantitative (not fully quantitative unless carefully controlled)
May not detect low-abundance proteins effectively
Agarose Gel Electrophoresis – Q&A Set
Q1. What does this setup demonstrate?
This setup demonstrates Agarose Gel Electrophoresis, a technique used to separate DNA (or RNA)
fragments based on their size using an electric field applied through a gel matrix.
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Q2. What is the principle of this technique?
DNA and RNA are negatively charged due to their phosphate backbone. When placed in an electric field,
they migrate towards the positive electrode (anode). Smaller fragments move faster through the pores of
the agarose gel than larger ones, enabling size-based separation.
Q3. What are the applications of this technique?
Checking the quality and quantity of DNA or RNA
Analyzing PCR products
Genotyping and mutation detection
Restriction fragment analysis
Preparative separation of nucleic acid fragments
Confirming plasmid digestion or ligation in cloning
Q5. What are the main steps of agarose gel electrophoresis?
Preparation of the agarose gel
Loading the DNA samples mixed with loading dye
Running the gel in a buffer system (usually TAE or TBE)
Electrophoresis under an electric current
Staining the gel with DNA-binding dyes (e.g., ethidium bromide or SYBR Safe)
Visualization under UV or blue light
Q6. Write the name of the organism from which agarose is isolated.
Agarose is isolated from red algae, primarily from species like Gelidium and Gracilaria.
Q7. Why is agarose used as the gel medium?
Agarose forms a porous matrix when solidified, allowing nucleic acids to move through it based on size.
It’s non-toxic, easy to prepare, and provides good resolution for DNA fragments between 100 bp to 25 kb.
Q8. What is the role of the loading dye?
The loading dye:
Adds density so the sample sinks into the well
Contains tracking dyes (e.g., bromophenol blue) that help monitor the progress of electrophoresis
Often contains glycerol or sucrose
Q9. How are DNA fragments visualized?
DNA is visualized by staining with intercalating dyes like ethidium bromide, SYBR Safe, or GelRed, and
then observed under UV transilluminator system.
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Q10. What factors affect the migration of DNA in the gel?
Size of DNA fragments
Concentration of agarose (higher % = smaller pore size)
Voltage applied
Type of buffer used
Conformation of DNA (supercoiled, linear, circular)
Q11. What are the limitations of this technique?
Lower resolution for very large or very small fragments
Cannot distinguish between sequences of same size
Not quantitative unless combined with additional tools
Use of ethidium bromide poses health and safety risks
Q12. How do you choose the concentration of agarose?
0.7% for large DNA fragments (>2 kb)
1.0–1.2% for general use (500 bp to 10 kb)
1.5–2.0% for small fragments (<500 bp)
Polyacrylamide Gel Electrophoresis (PAGE) – Q&A Set
Q1. What is the principle of polyacrylamide gel electrophoresis (PAGE)?
PAGE separates molecules, usually proteins or small nucleic acids, based on their size and charge by
passing them through a polyacrylamide gel matrix under an electric field.
Q2. What types of PAGE are commonly used?
SDS-PAGE (Sodium Dodecyl Sulfate-PAGE): for separating proteins by size
Native PAGE: for separating proteins by size, charge, and conformation
Urea-PAGE or Denaturing PAGE: for nucleic acid analysis, especially small RNAs
Q3. What is the role of SDS in SDS-PAGE?
SDS is an anionic detergent that denatures proteins and coats them with a uniform negative charge, so
their separation is based solely on molecular weight.
Q4. What are the applications of PAGE?
Protein purity analysis
Molecular weight estimation of proteins
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Protein-protein interaction studies (via native PAGE)
Western blotting (PAGE is the first step)
Separation and analysis of short nucleic acids (PAGE with urea)
Q5. What are the components of a polyacrylamide gel?
Acrylamide: forms the polymer matrix
Bis-acrylamide: cross-linking agent
TEMED and APS: initiate polymerization of acrylamide
The concentration of acrylamide determines pore size (higher % = smaller pores).
Q6. How is PAGE different from agarose gel electrophoresis?
PAGE is used for proteins and small nucleic acids, with high resolution
Agarose gels are better for larger nucleic acids (like genomic DNA or PCR products)
PAGE uses toxic monomers and requires careful handling, while agarose is safer and easier to prepare
MS Media Preparation in Plant Tissue Culture – Q&A Set
Q1. What is MS medium and who developed it?
MS (Murashige and Skoog) medium is a widely used nutrient medium for plant tissue culture, developed
by Toshio Murashige and Folke Skoog in 1962. It supports the growth and development of a wide range
of plant species in vitro.
Q2. What are the main components of MS medium?
Macronutrients (e.g., nitrogen, phosphorus, potassium, calcium, magnesium)
Micronutrients (e.g., iron, manganese, zinc, copper, boron)
Vitamins (e.g., thiamine, nicotinic acid, pyridoxine)
Carbon source (typically sucrose)
Plant growth regulators (auxins, cytokinins – added as needed)
Gelling agent (e.g., agar, if solid medium is required)
Q3. What is the role of sucrose in MS medium?
Sucrose acts as the carbon and energy source for the plant tissues, especially important since cultured
tissues may not photosynthesize efficiently in vitro.
Q4. What is the pH of MS medium and how is it adjusted?
The pH of MS medium is typically adjusted to 5.6–5.8 using NaOH or HCl before autoclaving, as an
appropriate pH is critical for nutrient availability and tissue health.
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Q5. What precautions must be taken before autoclaving the medium?
Ensure pH is adjusted before sterilization
Add heat-sensitive components (e.g., plant hormones, antibiotics) after autoclaving, using sterile filtration
Use clean, properly labeled containers
Avoid caramelization of sugars by not overheating
Q6. Why is MS medium considered a standard in plant tissue culture?
Because it provides a balanced supply of nutrients, promotes rapid growth and organogenesis, and is
suitable for a wide variety of plant species and tissue types.
Surface Sterilization, Sterilization & Inoculation – Q&A Set
Q1. Why is surface sterilization of explants important in plant tissue culture?
Surface sterilization removes microbial contaminants (bacteria, fungi) from the explant's surface, ensuring
aseptic conditions for successful culture initiation and growth.
Q2. What chemicals are commonly used for surface sterilization of explants?
70% ethanol (for 30 seconds to 1 minute)
Sodium hypochlorite (1–2% for 5–15 minutes)
Mercuric chloride (HgCl₂) (0.1% for 1–5 minutes – used with caution)
Followed by multiple rinses with sterile distilled water
Q3. What is the purpose of autoclaving in plant tissue culture?
Autoclaving is used to sterilize media, glassware, and tools by applying steam under pressure (121°C, 15
psi, 15–20 minutes), which kills all microbes and spores.
Q4. What precautions must be taken during the surface sterilization of explants?
Do not overexpose explants to sterilants to avoid tissue damage
Ensure thorough rinsing to remove residual sterilants
Work quickly to prevent desiccation
Perform under sterile conditions when transferring to medium
Q5. What is inoculation in plant tissue culture?
Inoculation refers to the transfer of the sterile explant onto the culture medium using sterile instruments in
a laminar airflow cabinet to maintain aseptic conditions.
Q6. What aseptic techniques are essential during inoculation?
Use of a laminar airflow hood
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Flaming tools (e.g., forceps, scalpels) after dipping in alcohol
Wearing sterile gloves and lab coats
Minimizing exposure of open containers to air
Disinfecting the work area with 70% ethanol
Isolation of Genomic DNA
Q.1 What is the function of CTAB in the DNA extraction procedure?
Answer: CTAB, or cetyltrimethylammonium bromide, is an ionic detergent used to lyse the cell and nucleus
membranes of plant cells during DNA extraction. It separates nucleic acids from other cellular components
by forming an insoluble complex with nucleic acids.
Q.2 Why is it crucial to use a mortar and pestle in the initial stage of DNA extraction?
Answer: Using a mortar and pestle, the cell and nucleus walls of the cauliflower curd are shattered. This
mechanical disruption aids in the liberation of the cellular contents, including genomic DNA, for subsequent
extraction.
Q.3 What function does EDTA play in the extraction buffer?
Answer: Chelating agent ethylenediaminetetraacetic acid (EDTA) is added to the extraction buffer. It bonds
to divalent ions, such as magnesium, which serve as cofactors for enzymes that degrade DNA. EDTA
prevents DNA degradation during the extraction procedure by removing these ions.
Q. 4 What role does NaCl play in the DNA extraction process?
Answer: Sodium chloride, or NaCl, is introduced to the extraction buffer in order to create an isotonic
environment. It aids in maintaining the osmotic equilibrium and structural integrity of the cell and nuclear
membranes. Neutralizing the negative charges on the phosphate groups of DNA, NaCl facilitates the
aggregation and precipitation of DNA molecules.
Q. 5 What role does Tris/HCl play in the extraction buffer?
Answer: Tris/HCl, also known as Tris(hydroxymethyl)aminomethane hydrochloride, is a buffering agent.
During the extraction process, it helps sustain a stable pH, which is crucial for DNA stability. Tris/HCl
maintains the optimal pH, which is typically mildly alkaline, for subsequent DNA purification procedures.
Q. 6 In the DNA extraction procedure, how are impurities separated from the CTAB-nucleic acid
complex?
Answer: Polysaccharides, phenolic compounds, proteins, and other cell lysates are extracted with
chloroform to remove impurities from the CTAB-nucleic acid complex. Proteins are denatured by
chloroform, which also facilitates the separation of the aqueous and organic phases. While the nucleic acids
partition into the organic phase, the impurities remain in the aqueous phase.
Q. 7 What is the purpose of the DNA extraction procedure's precipitate step?
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Answer: The DNA extraction procedure concludes with the precipitation phase. It requires extracting
nucleic acids from CTAB detergent. By treating the aqueous solution with a precipitation solution comprised
of CTAB and high-concentration NaCl, the nucleic acids precipitate while the detergent, which is more
soluble in alcohol, is removed. This step assists in the separation of nucleic acids from any remaining
contaminants and facilitates subsequent purification.
Q. 8 What measures must be taken to prevent DNA degradation during the extraction procedure?
Answer: Minimize the time between sample homogenization and the addition of the CTAB buffer to prevent
DNA degradation. This decreases DNA's exposure to prospective nucleases. In addition, the components of
the extraction buffer, such as EDTA and NaCl, inhibit the activity of DNA-degrading enzymes. Maintaining
a correct pH and working in a clean, sterile environment can also aid in DNA preservation.
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