Agarose and Polyacrylamide Gel Electrophoresis
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Questions and Answers

What is the main advantage of using TAE buffer in gel electrophoresis?

  • It contains borate which inhibits enzymes.
  • It works better for cloning experiments. (correct)
  • It has a higher buffering capacity than TBE.
  • It allows for a higher resolution of smaller fragments.

    • Agarose gels can only be used for small DNA molecules.

    False

    What is the effect of pH changes on nucleic acids during electrophoresis?

    Changes in pH affect the net charge of the nucleic acids.

    The average pore size of a 6% agarose gel is approximately ______ nm.

    <p>30</p> Signup and view all the answers

    Match the following gel properties with their corresponding gel type:

    <p>Agarose = Non-toxic and wider gaps Polyacrylamide = Moderately hazardous and complex TAE Buffer = Better for cloning TBE Buffer = Higher resolution for smaller fragments</p> Signup and view all the answers

    Which buffer would you use to achieve better separation of larger fragments (>3kb) during gel electrophoresis?

    <p>TAE</p> Signup and view all the answers

    Polyacrylamide gels are made up of multiple molecular types in contrast to agarose gels.

    <p>False</p> Signup and view all the answers

    What is the function of EDTA in running buffers?

    <p>EDTA prevents nuclease from degrading the nucleic acids.</p> Signup and view all the answers

    What is the primary role of Taq DNA Polymerase in PCR?

    <p>To amplify DNA by adding nucleotides</p> Signup and view all the answers

    Pfu DNA Polymerase is less accurate than Taq Polymerase.

    <p>False</p> Signup and view all the answers

    What is the typical total volume required for a PCR reaction?

    <p>15μL</p> Signup and view all the answers

    PCR requires two primers, the forward primer and the __________ primer.

    <p>reverse</p> Signup and view all the answers

    Match the DNA polymerases with their characteristics:

    <p>Taq DNA Polymerase = Heat-stable, suitable for standard PCR Pfu DNA Polymerase = High-fidelity, proofreads DNA High-fidelity PCR = Reduces errors during amplification Gel Electrophoresis = Separates DNA fragments by size</p> Signup and view all the answers

    Which property is NOT typical for primers used in PCR?

    <p>End with A/T pairs</p> Signup and view all the answers

    Gel electrophoresis can be used to analyze the results of a PCR reaction.

    <p>True</p> Signup and view all the answers

    What is the ideal G/C content range for PCR primers?

    <p>40-60%</p> Signup and view all the answers

    During high-fidelity PCR, the error rate is __________ compared to standard PCR.

    <p>lower</p> Signup and view all the answers

    Which of the following conditions can affect the efficiency of PCR?

    <p>All of the above</p> Signup and view all the answers

    Which enzyme is ideally used for PCR amplification of DNA fragments up to 5 kb in length?

    <p>Taq DNA Polymerase</p> Signup and view all the answers

    Pfu DNA polymerase is faster than Taq DNA polymerase.

    <p>False</p> Signup and view all the answers

    What is the optimal temperature for Taq polymerase to incorporate nucleotides?

    <p>72°C</p> Signup and view all the answers

    Pfu DNA polymerase is used for _____ PCR and primer-extension reactions.

    <p>high-fidelity</p> Signup and view all the answers

    Match the following DNA polymerases with their key features:

    <p>Taq DNA Polymerase = Thermostable, used for PCR Pfu DNA Polymerase = High-fidelity, slower than Taq DNA polymerase I = Requires a template, has exonuclease activity Klenow Fragment = Eliminates 5' to 3' exonuclease activity</p> Signup and view all the answers

    What is the rate at which Taq polymerase incorporates nucleotides?

    <p>1-2 kilobases per minute</p> Signup and view all the answers

    Phusion polymerase can amplify long templates at a rate of 4 kb per minute.

    <p>False</p> Signup and view all the answers

    What type of template does terminal transferase require for deoxynucleotide addition?

    <p>It does not require a template.</p> Signup and view all the answers

    TA cloning utilizes a vector that has single 5'-______ overhangs.

    <p>T</p> Signup and view all the answers

    What characteristic of DNA allows it to migrate towards the positively charged electrode during gel electrophoresis?

    <p>It is negatively charged.</p> Signup and view all the answers

    Taq polymerase has a proof-reading domain which reduces its error rate.

    <p>False</p> Signup and view all the answers

    What is the primary purpose of gel electrophoresis?

    <p>To separate mixtures of molecules by size.</p> Signup and view all the answers

    The backbone of DNA is negatively charged due to the presence of ______.

    <p>phosphate groups</p> Signup and view all the answers

    Match the following DNA polymerases with their characteristics:

    <p>Taq polymerase = Higher error rate, no proof-reading Pfu DNA Polymerase = Higher fidelity, slower speed Terminal Transferase = No template required for addition Phusion polymerase = High speed and fidelity</p> Signup and view all the answers

    What is the unique activity of Taq polymerase when it amplifies DNA?

    <p>It adds a single adenosine to the 3'-ends.</p> Signup and view all the answers

    Which component is essential for initiating DNA synthesis in PCR?

    <p>Primers</p> Signup and view all the answers

    Taq DNA Polymerase is known for its high fidelity when amplifying DNA.

    <p>False</p> Signup and view all the answers

    What is the main use of Gel Electrophoresis?

    <p>To separate DNA fragments based on size</p> Signup and view all the answers

    The ______ enzyme synthesizes new strands of DNA complementary to the target sequence in PCR.

    <p>DNA polymerase</p> Signup and view all the answers

    Match the DNA polymerases with their characteristics:

    <p>Taq DNA Polymerase = Fast amplification Pfu DNA Polymerase = High fidelity DNA polymerase I = Repair and replication Reverse Transcriptase = Converts RNA to DNA</p> Signup and view all the answers

    What is the purpose of nucleotides in the PCR process?

    <p>To act as building blocks for new DNA strands</p> Signup and view all the answers

    High-fidelity PCR uses polymerases that provide lower error rates than Taq polymerase.

    <p>True</p> Signup and view all the answers

    What type of DNA polymerase is typically used in high-fidelity PCR reactions?

    <p>Pfu DNA Polymerase</p> Signup and view all the answers

    During gel electrophoresis, DNA fragments move through the gel towards the ______ electrode.

    <p>positive</p> Signup and view all the answers

    What role do primers play in the PCR process?

    <p>They serve as starting points for DNA synthesis</p> Signup and view all the answers

    Study Notes

    Agarose Gel Electrophoresis

    • Agarose is a natural polysaccharide derived from seaweed.
    • Agarose gels are formed by dissolving agarose in boiling water and cooling the solution below 45°C.
    • The pore size of agarose gels depends on the agarose concentration.
    • 6% agarose has a pore size of approximately 30 nm.
    • 4% agarose has a pore size of 70 nm.
    • 2% agarose has a pore size of 150 nm.
    • Larger pore size agarose is used for electrophoresis of large DNA molecules.

    Polyacrylamide Gel Electrophoresis

    • Polyacrylamide is a synthetic polymer.
    • Polyacrylamide gels are considered moderately hazardous due to potential toxicity.
    • Polyacrylamide gels are made up of one large molecular type.
    • Polyacrylamide gels are poured vertically.

    Running Buffers

    • Running buffers are used to allow nucleic acids to move through the gel matrix.
    • Running buffers maintain pH and ion concentration during electrophoresis.
    • Running buffers contain EDTA to prevent nuclease degradation of nucleic acids.
    • TAE buffer is based on acetic acid and is better for DNA cloning.
    • TBE buffer is based on boric acid and is better for separating smaller fragments (< 3 kb).

    TA Cloning

    • TA cloning vectors have single 5'-T overhangs at each end.
    • TA cloning is a simple and efficient method for cloning PCR products.
    • Taq polymerase has non-template dependent activity that preferentially adds a single adenosine to the 3'-ends of DNA molecules.
    • PCR products amplified by Taq polymerase have single 3'-A overhangs.

    Gel Electrophoresis

    • Gel electrophoresis separates molecules based on size and charge.
    • DNA is negatively charged due to phosphate groups in nucleotides.
    • DNA migrates towards the positively charged electrode during electrophoresis.

    Components of PCR

    • DNA template: 104-107 molecules
    • Primers: 0.1–1 μM
    • Nucleotides (dNTPs): 50 μM
    • DNA polymerase: 0.5-2.5 units per 50 μl
    • DNA polymerase buffer (X buffer)
    • Sterile water

    PCR Principle

    • PCR requires three steps:
      • Denaturation: Separating double-stranded DNA into single strands.
      • Annealing: Primers binding to template strands.
      • Extension: DNA polymerase synthesizing new strands from primers.

    PCR Program

    • PCR is performed in a thermal cycler that controls temperature changes.
    • Each cycle consists of three steps: denaturation, annealing, and extension.
    • The number of cycles determines the amount of DNA amplification.

    Polymerases Commercially Available

    • DNA polymerase I:

      • Isolated from E. coli.
      • Has 5' to 3' DNA polymerase activity, 3' to 5' exonuclease activity, and 5' to 3' exonuclease activity.
      • Used for DNA synthesis from a single-strand DNA template at 37°C.

    • Klenow Fragment of DNA polymerase I:

      • Fragment of DNA polymerase I with 5' to 3' polymerase and 3' to 5' exonuclease activity, but no 5' to 3' exonuclease activity.
      • Used for DNA synthesis.

    • Taq DNA Polymerase:

      • Thermostable enzyme isolated from Thermus aquaticus.
      • Used for PCR amplification of DNA fragments up to 5 kb in length.
      • Used for DNA labeling and sequencing.
      • Ideal for TA cloning.
      • Adds a single adenosine to the 3'-ends of DNA molecules.

    • Pfu DNA polymerase:

      • Isolated from Pyrococcus furiosus.
      • Has 3' to 5' exonuclease activity.
      • Used for high-fidelity PCR and primer-extension reactions.

    • Phusion:

      • High-fidelity DNA polymerase.
      • Has 3' to 5' exonuclease activity.
      • Has a faster speed than Taq polymerase.

    Terminal Transferase

    • Mammalian enzyme expressed in lymphocytes.
    • Catalyzes deoxynucleotide addition to a free 3'-OH end without a template.
    • Used to generate DNA blunt ends and label DNA 3' ends.

    PCR Primer Design

    • Primers should generally have the following properties:
      • Length of 18-30 bases
      • 40-60% G/C content
      • End with G/C (GC clamp)
      • Start and end with 1-2 G/C pairs
      • Melting temperature (Tm) of 65-75°C
      • Primer pairs should have a Tm within 5°C of each other
      • Avoid regions of secondary structure, GC-rich, and AT-rich domains
      • Avoid runs of 4 or more of one base or dinucleotide repeats.

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    Related Documents

    PCR & Electrophoresis Lecture Notes PDF

    Description

    Explore the fascinating world of gel electrophoresis! This quiz covers the characteristics and preparation of agarose and polyacrylamide gels, as well as the important role running buffers play in the process. Test your knowledge on electrophoresis techniques and their applications in molecular biology.

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