Molecular Biology Techniques Quiz

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Questions and Answers

What is the primary factor that SDS-PAGE uses to separate proteins?

Molecular weight of the proteins (correct)

Shape of the proteins

Charge of the proteins

Concentration of the buffer

Which of the following factors does NOT affect electrophoresis?

pH and ionic strength of buffer

Electric field strength

Size and shape of the molecules

Color of the proteins (correct)

What is the purpose of Sodium Dodecyl Sulfate (SDS) in SDS-PAGE?

To alter the pH of the buffer

To increase the temperature of the gel

To denature proteins and provide a uniform charge (correct)

To enhance the staining of proteins

Which technique involves the use of filter paper for separation of small charged molecules?

Paper electrophoresis Signup and view all the answers

During electrophoresis, molecules migrate towards which pole based on their charge?

The positive pole Signup and view all the answers

What primarily affects the rate of migration of proteins in SDS-PAGE?

Molecular weight of the proteins Signup and view all the answers

What is the effect of temperature on electrophoresis?

Higher temperatures increase migration speed. Signup and view all the answers

Which statement about the results obtained from paper electrophoresis is accurate?

Separated components can be detected using various staining methods. Signup and view all the answers

What is the primary purpose of Southern blotting in molecular biology?

Detection of specific DNA sequences Signup and view all the answers

What is the first step in the Southern blotting process?

Digestion of genomic DNA with restriction enzymes Signup and view all the answers

Which component is crucial for the visualization of ethidium bromide during electrophoresis?

Ultraviolet light source Signup and view all the answers

In Northern blotting, what type of molecules are detected?

mRNA molecules Signup and view all the answers

Which step in Northern blotting follows the separation of RNA according to size?

Fixation of RNA to the solid matrix Signup and view all the answers

What is the function of the labeled probe in a Southern blot?

To hybridize with complementary sequences Signup and view all the answers

What technique is used to separate the RNA in a Northern blot before its transfer to a solid support?

Gel electrophoresis Signup and view all the answers

During the Southern blotting process, which method is employed to visualize the bands of DNA after hybridization?

Autoradiography Signup and view all the answers

What is the primary purpose of including bromophenol blue in the loading buffer?

To provide color for visual tracking Signup and view all the answers

When running an electrophoresis gel, where does DNA migrate towards?

To the anode (+) Signup and view all the answers

What is the significance of including a DNA ladder on the gel?

To determine the sizes of unknown DNAs Signup and view all the answers

What occurs when the power supply is turned on during gel electrophoresis?

Bubbles form on the electrodes Signup and view all the answers

How can ethidium bromide facilitate the visualization of DNA on a gel?

By fluorescing under UV light when bound to DNA Signup and view all the answers

What is a critical safety precaution when working with ethidium bromide?

Gloves should be worn at all times Signup and view all the answers

What size of DNA does bromophenol blue migrate at approximately the same rate as?

300 bp Signup and view all the answers

What is the purpose of allowing the gel to destain in water after staining?

To prevent over-staining of the gel Signup and view all the answers

What is the role of N,N’-methylene-bis-acrylamide in the formation of polyacrylamide gel?

It serves as a cross-linking agent. Signup and view all the answers

What initiates the polymerization process in the formation of polyacrylamide gel?

Ammonium persulfate. Signup and view all the answers

Which component is NOT included in the de-staining buffer for polyacrylamide gel?

Coomassie brilliant blue. Signup and view all the answers

How does the size of DNA fragments affect their migration rate in an agarose gel?

Smaller DNA fragments migrate faster than larger ones. Signup and view all the answers

What is the purpose of the 6X sample loading buffer when preparing DNA samples?

To increase the visibility of the samples in the gel. Signup and view all the answers

What charge does DNA carry, and how does this affect its movement in an electrical field?

Negatively charged; it moves towards the positive pole. Signup and view all the answers

What is a critical factor that can affect how fast DNA migrates through an agarose gel?

Strength of the electrical field. Signup and view all the answers

Which of the following correctly describes the relationship between DNA size and migration in gel electrophoresis?

Migration is inversely proportional to the log10 of DNA size. Signup and view all the answers

Study Notes

Biomedical Techniques CLSB-222

Course instructor: Dr. Ahmad Alamri, M.H.S, Ph.D.
Instructor contact: [email protected]

Electrophoretic Techniques

Electrophoresis is the migration of charged molecules in an electric field.
It's commonly used in clinical labs for isolating and quantifying serum proteins, isoenzymes, hemoglobin, and lipoproteins.
Factors affecting electrophoresis include the net electric charge on molecules (pH and ionic strength of buffer), size and shape of molecules, electric field strength, and the nature of the supporting media, and temperature of operation.

A) Paper Electrophoresis

A form of electrophoresis performed on filter paper.
Useful for separating small charged molecules like amino acids and small proteins.
Samples are spotted, and a high voltage is applied, separating molecules based on charge.
Separated components are detected by staining methods based on their chemical identity.

B) SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

A widely used technique in biochemistry, forensics, genetics, and molecular biology for separating proteins.
Primarily separates proteins based on their molecular weights.
SDS (Sodium Dodecyl Sulfate) denatures proteins, binds to hydrophobic regions, disrupting non-covalent bonds, and giving proteins a negative net charge.
Proteins then migrate through the gel based on their size, with smaller proteins moving faster.
The rate of migration during SDS-PAGE is determined by the molecular weight.

Polyacrylamide Gel

Formed by co-polymerization of acrylamide and N,N'-methylene-bis-acrylamide.
Polymerization requires ammonium persulfate and tetramethylene ethylene diamine (TEMED) as catalysts.

Staining the Gel

Staining the gel involves using a buffer containing glacial acetic acid, methanol, and Coomassie brilliant blue 250-R.
Destaining the gel uses a buffer containing glacial acetic acid and methanol to remove excess stain.
Ethidium bromide stains DNA, allowing visualization under UV light. Caution: Ethidium bromide is a powerful mutagen and is moderately toxic.

C) Agarose Gel Electrophoresis

Used to separate DNA fragments based on their size using a gel made of agarose.
Separates DNA by their rate of movement through the gel in an electric field.
Commonly used to determine the presence and size of PCR products.

Sample Preparation

Mix DNA samples with 6X sample loading buffer containing bromophenol blue (for color) and glycerol (for weight).
This improves visibility during loading and increases sample density for better placement in the gel wells.

Loading the Gel

Carefully place the pipette tip over a well and gently expel the sample.
The sample should sink into the well, avoiding puncturing the gel with the tip.

Running the Gel

Place the cover on the electrophoresis chamber, connecting the electrical leads to the power supply.
Ensure the leads are correctly attached (DNA migrates towards the anode, red).

DNA Ladder Standard

A DNA ladder containing DNA fragments of known sizes is included on the gel.
This allows for easier determination of unknown DNA sizes.
Bromophenol blue runs at approximately the same rate as a 300 bp DNA molecule.

Southern Blotting

Method for detecting specific DNA sequences in DNA samples.
- Digests genomic DNA with restriction enzymes.
- Separates DNA fragments by agarose gel electrophoresis.
- Denatures DNA fragments.
- Transfers DNA to a solid support (nylon or nitrocellulose).
- Hybridizes immobilized DNA to a labeled probe.
- Detects complementary probes (e.g., by autoradiography).
- Estimates the size and number of bands after digestion.

F) Northern Blotting

Method routinely used for detecting specific RNA sequences in RNA samples.
Isolates intact mRNA.
Separates RNA by size (using denaturing agarose gel).
Transfers RNA to a solid support.
Fixes RNA to the solid matrix.
Hybridizes with complementary probes.
Removes unbound probe molecules.
Detects, captures, and analyzes images of specifically bound probe molecules.

F) Western Blotting

"Immunoblotting" is a technique for determining the molecular weight and quantity of proteins in a complex mixture; it is also used in medical diagnostics.
Separates proteins by SDS-PAGE (polyacrylamide gel electrophoresis).
Transfers proteins to a membrane (e.g., nitrocellulose).
Uses primary and secondary antibodies to detect target proteins.
Detects target proteins using colorimetric or chemiluminescent probes.

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Description

Test your knowledge on key molecular biology techniques, including SDS-PAGE, Southern blotting, and Northern blotting. Answer questions regarding the principles and purposes of these techniques as well as the factors affecting protein and nucleic acid separation. This quiz covers essential concepts and methods used in the laboratory.