Instruction 6

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Questions and Answers

What is the primary purpose of adding a comb to the agarose solution during gel preparation?

To create a stable, hydrogen-bonded polymer network.

To form wells in the gel for loading DNA samples. (correct)

To cool the agarose solution rapidly

To facilitate the even distribution of the agarose solution.

Why might a higher concentration of agarose be selected for gel electrophoresis?

To decrease the temperature for gel solidification.

To increase the speed of DNA migration through the gel.

To separate very large DNA molecules with similar sizes.

To separate very small DNA molecules with similar sizes. (correct)

Which factor is primarily responsible for the stabilization of the agarose gel network?

Electrostatic interactions between agarose and the buffer.

Covalent bonds between agarose molecules.

The addition of a polymerizing factor during gel preparation.

Hydrogen bonds formed between agarose polymers. (correct)

What is the approximate temperature to which the agarose solution is typically cooled before pouring it into the gel form?

60°C Signup and view all the answers

Why is it essential to use the same buffer for dissolving the agarose and filling the electrophoresis chamber?

To maintain a stable pH and conductivity throughout the electrophoresis process. Signup and view all the answers

What is the primary factor determining the migration speed of DNA molecules through an agarose gel?

The size and mass of the DNA molecule Signup and view all the answers

Which characteristic of agarose makes it suitable for separating DNA fragments?

Its ability to create a cross-linked, porous structure Signup and view all the answers

How does increasing the concentration of agarose affect the gel's pore size?

It decreases the pore size Signup and view all the answers

What is a major disadvantage of using agarose gels in electrophoresis?

They have low mechanical resistance Signup and view all the answers

Why is it necessary for DNA to migrate through a gel matrix for effective separation, rather than just a solution?

To allow separation based on molecular size Signup and view all the answers

The migration speed of linear double-stranded DNA molecules in an agarose gel is reversely proportional to which value?

The log10 of the molecular mass Signup and view all the answers

If you wanted to separate large protein molecules, which electrophoresis method would be most appropriate?

Agarose gel electrophoresis Signup and view all the answers

Besides the size of the DNA molecule, what other factor impacts the relative mobility of DNA in electrophoresis?

The ionic strength of the buffer Signup and view all the answers

What is the optimal agarose concentration for separating DNA molecules in the range of 0.5 - 7 kbp?

0.9% Signup and view all the answers

If a DNA fragment has a size of 15 kbp and is migrating through an agarose gel, which of the following is NOT a direct effect of using too high a voltage?

Faster migration of linear dsDNA molecules Signup and view all the answers

Which of these is NOT a factor that is responsible for effective separation of nucleic acids during agarose gel electrophoresis?

Maintaining a neutral temperature Signup and view all the answers

What is the recommended voltage range for standard gels during electrophoresis?

5-8 V/cm Signup and view all the answers

What is a common consequence of using too low a voltage during agarose gel electrophoresis?

Diffusion and widening/dispersion of bands Signup and view all the answers

What is the general concentration range for buffers, such as Tris/Acetate/EDTA (TAE) and Tris/Borate/EDTA (TBE) during electrophoresis?

50 mM Signup and view all the answers

What is a practical difference between TAE and TBE buffers as described in the text?

TAE is less stable than TBE Signup and view all the answers

Which of the following is NOT significantly affected by the salt concentration in electrophoretic buffers?

Gel purity Signup and view all the answers

What is the primary principle underlying the movement of molecules during electrophoresis?

The movement of charged molecules in an electric field. Signup and view all the answers

What is the term for the movement of negatively charged molecules towards the positive electrode (anode) in electrophoresis?

Anaphoresis Signup and view all the answers

Why is electrophoresis in a free solution less effective for separation compared to using an electrophoretic medium?

Free solutions lack the ability to stabilize the electrolyte. Signup and view all the answers

What is a crucial property that a good electrophoretic medium should possess?

It should not interact with molecules, ensuring separation based on physical properties only. Signup and view all the answers

How does the concentration of an electrophoretic medium typically affect the separation ability of the technique?

It regulates the size of the pores and the range of molecule separation. Signup and view all the answers

What role does the use of porous media play in gel electrophoresis?

It allows additional fractionation based on molecular sieve principle. Signup and view all the answers

Which of the following is the most typical material of a gel used in gel electrophoresis?

Agarose Signup and view all the answers

What is agarose, the material often used as a medium in electrophoresis?

A natural polysaccharide obtained from edible agar Signup and view all the answers

What is the primary purpose of using a loading buffer in DNA electrophoresis?

To increase the density of the sample and allow it to be loaded into the gel wells Signup and view all the answers

Which of the following DNA conformations migrates the fastest during gel electrophoresis?

Supercoiled Signup and view all the answers

What is the role of the molecular weight marker (M) in gel electrophoresis?

To estimate the sizes of the DNA fragments in the sample Signup and view all the answers

Which of the following describes the relationship between the migration speed of linear DNA and its molecular mass during electrophoresis?

Migration speed is inversely proportional to the log10 of the molecular mass. Signup and view all the answers

What is the primary function of bromophenol blue and xylene cyanol in a loading buffer?

To follow the progression of electrophoresis. Signup and view all the answers

A plasmid with a break in one strand of its DNA is known as:

Nicked circle (OC) Signup and view all the answers

When would TBE buffer be most suitable over TAE buffer for DNA electrophoresis?

When high resolution is needed, especially for long runs. Signup and view all the answers

Which of the following DNA forms migrates the slowest in a gel?

Nicked Circle Signup and view all the answers

What is the primary purpose of using a dye in gel electrophoresis?

To make the DNA bands visible for observation and analysis. Signup and view all the answers

Why are dyes that migrate faster than DNA used in electrophoresis?

To allow the tracking of the DNA front and determining when electrophoresis is complete. Signup and view all the answers

Which of the following is a major concern associated with ethidium bromide?

It is a mutagenic and carcinogenic substance, posing health risks. Signup and view all the answers

What is a primary benefit of using dyes like SYBR green and Midori Green over ethidium bromide?

They have lower toxicity and similar or greater sensitivity, than ethidium bromide. Signup and view all the answers

When illuminating with UV light, what causes DNA bands to be visible after dyeing?

The dye fluoresces, emitting light that makes the DNA bands visible. Signup and view all the answers

Besides fluorescent dyes, what other method can be used to achieve very high sensitivity in detecting DNA bands?

Using radioisotopes. Signup and view all the answers

What is a common documentation method for polyacrylamide gel electrophoresis?

Allowing the gel to dry completely. Signup and view all the answers

What information can be derived from staining the bands on a electrophoresis gel?

The distance of migration and their amount in a band or spot. Signup and view all the answers

What is the fundamental mechanism by which an electrophoretic medium enhances the separation of molecules?

It acts as a molecular sieve, impeding the progress of larger molecules. Signup and view all the answers

Which of the following best describes the relationship between the concentration of an electrophoretic medium and its effect on molecular separation?

Changes in concentration allow for the regulation of pore size to select the range of molecule separation. Signup and view all the answers

Given that agarose is derived from agar, what is its fundamental chemical nature that makes it suitable for electrophoresis?

It is a non-ionic polysaccharide with galactose derivatives providing the polymer structure. Signup and view all the answers

Why is it crucial that the electrophoretic medium does not chemically interact with the molecules being separated?

Chemical interactions would alter the physical properties of the molecules, such as their size or shape. Signup and view all the answers

If electrophoresis is performed in a free liquid solution, what is the primary reason that makes it ineffective for separation?

The absence of a porous medium does not allow for size-based separation. Signup and view all the answers

In the context of electrophoresis, what is the significance of a 'colloidal solution' as mentioned in the text?

It is a solution where particles are suspended, but not dissolved, allowing molecules with charge to move within it. Signup and view all the answers

What is the fundamental difference between anaphoresis and cathaphoresis?

In anaphoresis the charge of molecules of interest is negative, while in cathaphoresis its positive. Signup and view all the answers

What primary role does the electrolyte play within an electrophoretic medium?

It functions to stabilize and buffer the pH within the medium to enhance separation. Signup and view all the answers

What is the maximum recommended voltage for performing electrophoresis as per standard guidelines?

100-120 V Signup and view all the answers

If the DNA fragment separation appears unclear after the initial electrophoresis, what should be done next?

Re-run the gel in the electrophoresis apparatus for a few more minutes Signup and view all the answers

What component of the gel electrophoresis process helps visualize DNA after separation?

Gel Doc EZ System Signup and view all the answers

What should be included in the report regarding the PCR sample preparation?

Concentration and purity ratios of the samples Signup and view all the answers

How should deviations from experimental protocols be documented in the report?

With specific explanations about potential causes Signup and view all the answers

What is a key purpose of using a loading buffer in electrophoresis?

To facilitate tracking of the DNA migration Signup and view all the answers

What essential information does the report require concerning DNA sample isolations?

Concentration in ng/µg and purity absorbency ratios Signup and view all the answers

Which technique can be used to assess the performance and results of PCR in electrophoresis?

Evaluating DNA visualization in the Gel Doc EZ System Signup and view all the answers

Which DNA form migrates the fastest during electrophoresis?

Covalently closed circular (CCC) Signup and view all the answers

Which agarose concentration is ideal for separating DNA fragments in the range of 1 to 20 kbp?

0.6% (w/v) Signup and view all the answers

What is the purpose of adding a molecular weight marker in gel electrophoresis?

To compare fragment sizes against known standards Signup and view all the answers

What happens when the voltage in electrophoresis is set higher than the recommended level?

The fragments over 12-15 kbp may become smudged. Signup and view all the answers

Which factor primarily influences the mobility of DNA in an electrophoretic gel?

Composition and ionic strength of the buffer. Signup and view all the answers

How does the migration speed of linear DNA fragments in an agarose gel correlate with their molecular mass?

Reversely proportional to the log10 of molecular mass Signup and view all the answers

What effect does using a higher concentration of agarose have on DNA migration?

Decreases the pore size of the gel Signup and view all the answers

What is the effect of using buffers without adequate salt concentrations?

Minimal conductivity causing no movement of DNA. Signup and view all the answers

How does increasing agarose concentration specifically impact the size range of DNA that can be optimally separated?

It limits the separation to smaller DNA fragments. Signup and view all the answers

Which substances are typically found in the loading buffer used for gel electrophoresis?

Ficoll, glycerol, and sucrose Signup and view all the answers

During electrophoresis, what determines the difference in DNA migration rates?

The size and conformation of the DNA fragments Signup and view all the answers

What is a notable disadvantage of using TAE buffer over TBE buffer during electrophoresis?

TAE is less stable than TBE. Signup and view all the answers

What type of DNA structure is created from a plasmid with a break in one strand?

Nicked circles Signup and view all the answers

At what current intensity should standard gels typically operate during electrophoresis?

75 mA Signup and view all the answers

What is the role of the buffer’s pH in agarose gel electrophoresis?

It affects the migration speed of DNA molecules. Signup and view all the answers

What is the role of dyes such as bromophenol blue in the loading buffer?

They provide a visual means to track DNA during electrophoresis Signup and view all the answers

What effect does the size of DNA molecules have on their migration speed in agarose gel electrophoresis?

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

Which factor does NOT impact the relative mobility of DNA during electrophoresis?

DNA sequence composition Signup and view all the answers

What is a primary disadvantage of using agarose gels for electrophoresis?

Low mechanical resistance of agarose gels Signup and view all the answers

How does agarose concentration influence the properties of the gel?

Higher concentration creates smaller pores that slow down DNA migration. Signup and view all the answers

In the context of DNA electrophoresis, what does 'reversely proportional' mean in relation to migration speed?

Larger molecules have slower migration speeds. Signup and view all the answers

What characteristic makes agarose a non-toxic medium for gel electrophoresis?

It contains no harmful chemicals. Signup and view all the answers

Which of the following accurately describes the relationship between electrical current and DNA migration in agarose gel?

Increased current enhances separation of molecules of different sizes. Signup and view all the answers

What role does pH play in the migration of DNA during electrophoresis?

Adjusting pH changes the charge of DNA molecules. Signup and view all the answers

What is the maximum storage period for dried gel made from tissue paper or cellophane?

Infinite period Signup and view all the answers

What is the benefit of photographing an agarose gel after electrophoresis?

It documents the separation results of the DNA fragments. Signup and view all the answers

Which piece of equipment is NOT needed for performing DNA electrophoresis?

Electric balance Signup and view all the answers

How should the agarose solution be treated to ensure proper gel preparation?

Boiled in the microwave multiple times Signup and view all the answers

What is a crucial step in preparing agarose gels for electrophoresis?

Calculating the mass of agarose based on the desired gel concentration Signup and view all the answers

Which type of dye is used in the agarose gel preparation according to the document?

Midori Green Advance DNA Stain Signup and view all the answers

What must be ensured to avoid boiling over the agarose solution in the microwave?

Monitoring the temperature carefully Signup and view all the answers

Which of the following concentrations of agarose is NOT mentioned for preparing the gels?

0.8% Signup and view all the answers

Study Notes

DNA Electrophoresis

Electrophoresis is an analytical and sometimes preparative technique used in chemistry and molecular biology to separate molecules based on size and charge.
It involves the movement of charged molecules in an electric field, towards electrodes with opposite charges.
Molecules migrate towards positive (anode) for negatively charged particles and towards negative (cathode) for positively charged particles.
Electrophoresis in free solutions is less effective due to low resolution.
Electrophoretic media improve separation
- Stability
- Repeatability
- Resolution
These media can include tissue paper, cellulose nitrate, agarose, or polyacrylamide.
Porous media improve separation by acting as a molecular sieve.
Agarose and polyacrylamide are commonly used.
Agarose is a natural polysaccharide (polymer of galactose derivatives).
It produces a reversible gel at room temperature.
Agarose concentration affects the range of DNA molecules that can be separated effectively.
TAE and TBE buffers are commonly used for DNA electrophoresis; TAE is cheaper, but TBE is more stable.
Buffer composition/ionic strength is a factor affecting DNA migration.
Safe/low toxicity dyes like SYBR Green or Midori Green are used for visualization purposes.

Agarose Electrophoresis Advantages

Separates large protein molecules
Wide range of DNA fragments (hundreds of base pairs to 40 kbps) in a stable electric field.
Gel preparation is feasible.
Non-toxic

Agarose Electrophoresis Disadvantages

Low mechanical resistance
Low resolution
Relatively expensive

DNA Electrophoresis in Agarose

DNA molecules have a negative charge and migrate towards the positive electrode (anode).
Migration speed is inversely proportional to the logarithm of molecular mass.
Factors affecting relative mobility include:
- Agarose concentration
- Voltage
- Buffer composition/ionic strength
- Spatial conformation of DNA molecules
High voltage can lead to smudging of DNA bands.
DNA molecule diffusion can lead to widening of DNA bands in the gel.

Agarose Preparation and Electrophoresis

Agarose dissolves in water when heated to boiling.
It turns into a gel when cooled.
The gel is placed between electrodes in a chamber filled with buffer.
Gel concentration affects the range of molecules separated effectively.
The gel is loaded with samples and subjected to an electric field to separate DNA fragments
Agarose concentration can be adjusted to control the size of the pores, which allows regulation of separation range.

Dyeing DNA in Gel Electrophoresis

DNA molecules are not visible to the naked eye after electrophoresis.
Dyes are used for visualization.
Ethidium bromide was a common dye but has a mutagenic and carcinogenic nature; currently less used.
Alternatives exist with lower toxicity, such as SYBR Green or Midori Green.
Radioisotopes can also be used, but proper safety measures are needed.

Electrophoretic Separations Documentation

Separations in polyacrylamide gels can be documented by drying the gel.
Agarose gels cannot be dried directly, so photographs are used to document them.

Performing DNA Electrophoresis

Appropriate instruments, buffers, and reagents needed.
Techniques for agarose gel preparation, loading DNA samples, and carrying out electrophoresis.
Measuring DNA masses using markers.
Procedures for data collection.
Correct use of DNA electrophoresis instruments and appropriate sample preparation for electrophoresis.
Using a molecular weight marker for measuring the size of DNA fragments
Proper placement of samples in the wells
Controlling voltage and current

DNA Electrophoresis Report

Introduction about the procedures.
Reagents/equipment used and process descriptions
Results from DNA sample isolation with concentration values
Interpretation of electrophoresis results.
Include any variations or errors and appropriate explanations
Discuss the results of DNA separation in light of the experiment's objectives.
The report should contain an illustration from the electrophoresis.

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Description

This quiz covers the fundamentals of DNA electrophoresis, an essential technique in molecular biology and chemistry. Participants will explore how charged molecules are separated based on size and charge through different media. Understand the principles of agarose and polyacrylamide gels and their applications in DNA analysis.