DNA Electrophoresis Techniques 6

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Questions and Answers

Which of the following is NOT a reason why electrophoretic media are used in electrophoresis?

  • Chemical fractionation of molecules (correct)
  • Stability
  • Resolution
  • Repetition

What is the primary purpose of an electrophoretic medium in electrophoresis?

  • To provide a stable environment for the electrophoresis process (correct)
  • To act as a source of electric current
  • To facilitate the binding of DNA to the electrodes
  • To chemically modify the DNA molecules

Which of the following is NOT a characteristic of an ideal electrophoretic medium?

  • It should be able to chemically modify the molecules being separated (correct)
  • It should be able to be easily modified to change pore size
  • It should be inert to the molecules being separated
  • It should be able to stabilize the electrolyte solution

In electrophoresis, why does a negatively charged molecule migrate towards the anode?

<p>Because it is attracted to the positive charge of the anode (B)</p> Signup and view all the answers

What is the primary factor that determines the speed of migration of a DNA molecule in an electrophoretic medium?

<p>The molecular weight of the DNA molecule (A)</p> Signup and view all the answers

Which of the following is a commonly used gel for DNA electrophoresis?

<p>Agarose (C), Polyacrylamide (D)</p> Signup and view all the answers

What is the source of agarose used in electrophoresis?

<p>Purified edible agar (C)</p> Signup and view all the answers

What factor primarily stabilizes the network of polymers within an agarose gel?

<p>Hydrogen bonds between agarose molecules (B)</p> Signup and view all the answers

Which of the following statements is TRUE regarding the use of agarose gels for DNA separation?

<p>The concentration of agarose used directly influences the pore size of the gel matrix. (D)</p> Signup and view all the answers

Why is a comb used during the preparation of an agarose gel?

<p>To create wells for loading DNA samples into the gel (A)</p> Signup and view all the answers

Which of the following buffers is commonly used in agarose gel electrophoresis?

<p>Tris-acetate-EDTA (TAE) (A)</p> Signup and view all the answers

Why is it necessary to cool the agarose solution to approximately 60 degrees Celsius before pouring it into the mold?

<p>To ensure proper gel solidification (A)</p> Signup and view all the answers

How long does it typically take for an agarose gel to solidify after pouring?

<p>15-20 minutes (C)</p> Signup and view all the answers

What is the primary purpose of the electrophoretic buffer used in agarose gel electrophoresis?

<p>To provide a conductive medium for DNA migration (D)</p> Signup and view all the answers

During gel electrophoresis, what determines the direction of DNA migration?

<p>The charge of the DNA molecules and the direction of the electric field (D)</p> Signup and view all the answers

Why is it important to prevent the formation of air bubbles in the agarose gel?

<p>Air bubbles can impede DNA migration during electrophoresis (C)</p> Signup and view all the answers

Why is it essential to use the same buffer that the gel was cast in for the electrophoresis chamber?

<p>To maintain the pH of the gel throughout electrophoresis (B)</p> Signup and view all the answers

Which of the following statements is NOT true regarding the effect of agarose concentration on DNA separation in electrophoresis?

<p>A 0.3% agarose gel is ideal for separating DNA fragments between 1-20 kbp. (B)</p> Signup and view all the answers

What is the primary reason for the smudging of DNA fragments larger than 12-15 kbp when using high voltage in electrophoresis?

<p>Increased DNA degradation due to excessive heat. (D)</p> Signup and view all the answers

What is the most likely consequence of using a low voltage during DNA electrophoresis?

<p>Wider/more diffused bands, reducing resolution. (A)</p> Signup and view all the answers

Which of the following correctly describes the relationship between the voltage applied and the migration speed of DNA molecules in electrophoresis?

<p>Migration speed is directly proportional to the voltage applied. (C)</p> Signup and view all the answers

What is the main purpose of using buffers in DNA electrophoresis?

<p>To provide a stable pH environment for DNA separation. (C)</p> Signup and view all the answers

Why are Tris/Acetate/EDTA (TAE) and Tris/Borate/EDTA (TBE) commonly used buffers for DNA electrophoresis?

<p>They are inexpensive and readily available. (D)</p> Signup and view all the answers

What is the significance of the 'smiling' effect observed in DNA bands during electrophoresis?

<p>It suggests that the voltage applied is too high, causing uneven heating. (B)</p> Signup and view all the answers

If no salts were present in the electrophoresis buffer, what would be the most likely outcome?

<p>DNA molecules would not migrate at all. (D)</p> Signup and view all the answers

Which of the following factors would NOT influence the electrophoretic mobility of DNA in agarose gels?

<p>Length of the DNA molecule. (A)</p> Signup and view all the answers

What is the main advantage of using TBE buffer over TAE buffer for DNA electrophoresis?

<p>TBE buffer provides better resolution and stability during electrophoresis. (C)</p> Signup and view all the answers

Given the relationship between agarose concentration and pore size, which of the following statements is true?

<p>Lower concentrations result in a denser network of agarose, leading to smaller pores suitable for separating larger DNA fragments. (C)</p> Signup and view all the answers

What is the primary characteristic that determines the migration speed of DNA molecules during electrophoresis in an agarose gel?

<p>The size of the DNA molecule. (A)</p> Signup and view all the answers

Which of these factors does NOT directly influence the relative mobility of DNA during agarose gel electrophoresis?

<p>The temperature of the buffer solution. (C)</p> Signup and view all the answers

Why is agarose gel electrophoresis generally not optimal for separating very small DNA fragments (less than 100 bp)?

<p>The pores in agarose gels are too large for small fragments to be effectively separated. (B)</p> Signup and view all the answers

What is the relationship between the migration speed of linear dsDNA molecules and the number of base pairs?

<p>The migration speed is inversely proportional to the number of base pairs. (D)</p> Signup and view all the answers

Which of the following is NOT an advantage of agarose gel electrophoresis for separating DNA?

<p>High resolution of separation for even small DNA fragments. (C)</p> Signup and view all the answers

What is the primary reason why DNA molecules migrate towards the positive electrode (anode) during electrophoresis?

<p>The attractive force of the positive electrode (anode) due to the negative charge of DNA. (A)</p> Signup and view all the answers

How does the size of the DNA molecule affect its migration speed through an agarose gel?

<p>Smaller DNA molecules migrate faster because they encounter less resistance from the gel matrix. (D)</p> Signup and view all the answers

What is the purpose of using a buffer solution during agarose gel electrophoresis?

<p>The buffer solution conducts the electric current and maintains the pH of the gel system. (C)</p> Signup and view all the answers

Which of the following statements accurately describes the purpose of adding Midori Green dye to the dissolved agarose?

<p>Midori Green dye is a fluorescent stain that allows for visualization of the DNA fragments during electrophoresis. (C)</p> Signup and view all the answers

Which of the following statements accurately describes the purpose of the DNA ladder in electrophoresis?

<p>The DNA ladder acts as a positive control, verifying that the electrophoresis process is functioning correctly. (A)</p> Signup and view all the answers

What is the primary reason for the need to stabilize the pipette tip with a finger when loading DNA samples onto the gel?

<p>To prevent the pipette tip from vibrating and damaging the well in the gel. (C)</p> Signup and view all the answers

Why is it important to only partially insert the pipette tip into the well when loading DNA samples?

<p>To avoid puncturing the well and losing the sample. (D)</p> Signup and view all the answers

What is the reason for using a buffer in the electrophoresis apparatus?

<p>The buffer helps to maintain a constant pH environment and ionic strength for the electrophoresis process. (A)</p> Signup and view all the answers

Flashcards

Electrophoresis

A technique where charged molecules move in an electric field through a medium.

Anaphoresis

Movement of negatively charged molecules towards the positive electrode (anode).

Cathaphoresis

Movement of positively charged molecules towards the negative electrode (cathode).

Electrophoretic Medium

Substance that ensures stability and resolution in electrophoresis.

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Gel Electrophoresis

A method using agarose or polyacrylamide as a medium to separate macromolecules.

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Agarose

A natural polysaccharide used as an electrophoretic medium in gel electrophoresis.

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Molecular Sieve Principle

Separation of molecules based on size using porous media in electrophoresis.

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TAE/TBE buffer

Running buffers used to dissolve agarose for electrophoresis.

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Comb

A tool used to create wells in agarose gel for loading DNA samples.

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Cooling of agarose

The process of agarose gel solidifying after pouring.

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Molecular size separation

Separation of DNA molecules in electrophoresis based on size.

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Agarose concentration

The amount of agarose used in the gel, which affects separation ability.

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200 bp and 300 bp

Examples of small DNA fragments needing higher agarose concentration.

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5000 bp and 6000 bp

Examples of larger DNA fragments needing lower agarose concentration.

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Hydrogen bonds in agarose

Forces that stabilize the polymer network in the gel.

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Midori Green dye

A dye used to visualize DNA in agarose gels.

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DNA ladder

A molecular weight marker used in electrophoresis to estimate DNA fragment sizes.

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PCR Mix Plus Green

A pre-prepared mixture for PCR that includes a green dye for visual tracking.

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Loading buffer

A solution added to DNA samples to help visualize them during gel loading.

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Pore size regulation

Pore size in agarose gel can be altered by varying agarose concentration; higher concentrations yield smaller pores.

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Advantages of agarose electrophoresis

Agarose allows separation of large proteins and a wide range of DNA fragment sizes.

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DNA migration in electrophoresis

DNA moves towards the positive electrode due to its negative charge at neutral pH.

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DNA fragment size range

Agarose gel can separate DNA fragments from 100 bp to 40 kbp.

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Impact of agarose on DNA mobility

The size of DNA molecules affects their migration speed through agarose gel; larger molecules move slower.

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Factors affecting DNA mobility

DNA mobility in agarose electrophoresis is influenced by agarose concentration, current voltage, and buffer composition.

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Reversibility of agarose gel

Agarose gel formation is a reversible process; it can be dissolved back into a solution.

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Disadvantages of agarose electrophoresis

Agarose gels have low mechanical resistance, low resolution, and can be relatively expensive.

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Agarose concentration (0.3% w/v)

Can separate DNA molecules in the range of 5-60 kbp.

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Agarose concentration (0.6% w/v)

Can separate DNA molecules in the range of 1-20 kbp.

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Agarose concentration (1.2% w/v)

Can separate DNA molecules in the range of 0.4-6 kbp.

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Voltage in electrophoresis

Higher voltage quickens DNA migration but can smudge large fragments.

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Recommended voltage range

Should not exceed 5-8 V/cm in standard gels to avoid issues.

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Electrophoretic mobility

Depends on DNA composition and ionic strength of buffers.

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Importance of buffer in electrophoresis

Buffers maintain appropriate pH essential for DNA movement.

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Common buffers for DNA

TAE (Tris/Acetate/EDTA) and TBE (Tris/Borate/EDTA) are widely used.

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TAE vs. TBE

TAE is cheaper but less stable compared to TBE.

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Effect of low voltage

Results in slow migration and diffusion of DNA bands.

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Study Notes

DNA Electrophoresis

  • Electrophoresis is a technique used to separate charged molecules (like DNA) in a liquid solution using an electric field.
  • Molecules move towards the electrode with the opposite charge.
  • Negative molecules move towards the anode (positive electrode); this is called anaphoresis.
  • Positive molecules move towards the cathode (negative electrode); this is called cataphoresis.
  • In a free solution, separation is less effective.
  • Electrophoresis with a medium (e.g., agarose) improves resolution and stability.
  • Electrophoretic media like agarose or polyacrylamide stabilize electrolytes and separate macromolecules.
  • Agarose is a natural polysaccharide and is made from agar.
  • Agarose gels have pores that control the size of molecules that can move through allowing for separation based on size.

Agarose Electrophoresis Advantages

  • Separates large protein molecules.
  • High range of DNA fragments (from several hundred base pairs to 40 kb).
  • Feasibility of gel preparation.
  • Non-toxic.

Agarose Electrophoresis Disadvantages

  • Low mechanical resistance.
  • Low resolution.
  • Relatively expensive.

Factors Affecting DNA Migration

  • Agarose concentration: Higher concentration creates smaller pores, slowing down larger molecules.
  • Voltage: Higher voltage increases speed but can also smudge larger fragments.
  • Buffer composition/ionic strength: Affects sample movement and resolution.
  • DNA conformation: Affects rate of migration due to shape/size.

Agarose Gel Preparation/Procedure

  • Agarose is dissolved in a buffer (e.g., TAE or TBE) at specific concentrations (e.g., 1%, 2.5%) to control pore size.
  • The solution is poured into a casting tray and allowed to solidify, forming a gel.
  • Sample DNA is mixed with loading dye (with glycerol, etc) to increase density. Samples and a marker are loaded into the wells.
  • Electrophoresis is run at a specific voltage/current.
  • Migration of DNA through the gel is visible.

Documentation/Dyeing

  • DNA isn't visible in normal light, so dyes are needed.
  • Historically, ethidium bromide was used, but it is mutagenic and carcinogenic.
  • Alternatives like SYBR green or Midori Green are safer and often more sensitive.

DNA Electrophoresis Practice Procedure

  • Prepare agarose gel.
  • Prepare DNA samples.
  • Load samples into gel wells.
  • Run electrophoresis.
  • Visualize DNA bands with the appropriate dye.

DNA Conformations

  • Circular (supercoiled, open-circular, nicked).
  • Linear.

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Exercise No. 6 DNA Electrophoresis PDF

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