Molecular Biology: PCR and Electrophoresis Quiz
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Questions and Answers

What is the main purpose of PCR in molecular biology?

  • To convert DNA into mRNA
  • To sequence RNA directly
  • To isolate proteins
  • To exponentially amplify DNA (correct)

    • Which enzyme is primarily used in the PCR process?

  • Taq DNA Polymerase (correct)
  • RNA polymerase
  • DNA ligase
  • Reverse transcriptase

    • At what temperature does the denaturation phase of the PCR cycle occur?

  • 50-60 ºC
  • 37 ºC
  • 68-72 ºC
  • 94 ºC (correct)

    • Which of the following is NOT an application of PCR?

    <p>Protein synthesis</p> Signup and view all the answers

    What is the typical temperature range for the annealing phase in PCR?

    <p>50-60 ºC</p> Signup and view all the answers

    Who developed the PCR technique and won the Nobel Prize for it?

    <p>Kary Mullis</p> Signup and view all the answers

    What is the role of primers in the PCR process?

    <p>They provide a starting point for DNA synthesis</p> Signup and view all the answers

    Which application of PCR is specifically used for identifying genetic matching?

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

    What is the primary reason for wearing protective eyewear when observing DNA on a transilluminator?

    <p>To protect against UV light damage</p> Signup and view all the answers

    How does agarose concentration affect DNA fragment mobility?

    <p>Higher concentrations allow resolution of small fragments</p> Signup and view all the answers

    What might happen if water is mistakenly used instead of buffer in gel electrophoresis?

    <p>There will be no migration of DNA</p> Signup and view all the answers

    Which buffer is commonly utilized for duplex DNA fragment migration?

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

    What happens to DNA migration if a high voltage is applied during electrophoresis?

    <p>Migration accelerates</p> Signup and view all the answers

    Which statement is true regarding low versus high agarose concentrations?

    <p>Low concentrations allow better resolution for larger fragments</p> Signup and view all the answers

    What could occur if a concentrated buffer, like a 10X solution, is used during electrophoresis?

    <p>Heat generation that could melt the gel</p> Signup and view all the answers

    What additional step is necessary to quantify DNA using a spectrophotometer after a crude extraction?

    <p>Diluting the sample</p> Signup and view all the answers

    What is the primary function of the proofreading activity of DNA polymerase?

    <p>To correct nucleotide incorporation errors</p> Signup and view all the answers

    Which statement about Phusion High-Fidelity DNA Polymerase is accurate?

    <p>It was engineered by fusing a non-specific dsDNA-binding domain.</p> Signup and view all the answers

    What is the significance of maintaining equal concentrations of each dNTP in PCR?

    <p>It minimizes misincorporation levels.</p> Signup and view all the answers

    Which component in the PCR buffer helps promote primer annealing to the template DNA?

    <p>Potassium (K+) from KCl</p> Signup and view all the answers

    What role do magnesium ions (Mg2+) play in the PCR process?

    <p>They form complexes with dNTPs and primers.</p> Signup and view all the answers

    What pH range is typically suitable for the activity of DNA polymerase in a PCR buffer?

    <p>8.0 to 9.5</p> Signup and view all the answers

    Which PCR component can help enhance specificity by destabilizing weak hydrogen bonds?

    <p>Ammonium sulphate (NH4SO4)</p> Signup and view all the answers

    Which of the following statements about Taq DNA polymerase is incorrect?

    <p>It has a proofreading function.</p> Signup and view all the answers

    What is the primary method used for quantitating DNA using gel electrophoresis?

    <p>Comparing fluorescence intensity with DNA standards</p> Signup and view all the answers

    Which wavelength of UV light is primarily used for measuring nucleic acid concentration?

    <p>260 nm</p> Signup and view all the answers

    What indicates a pure DNA sample when using the OD260/OD280 ratio?

    <p>1.8</p> Signup and view all the answers

    What limitation does a regular Spectrophotometer have regarding DNA quantitation?

    <p>It cannot quantitate low amounts of DNA (less than 250 ng/mL).</p> Signup and view all the answers

    What does an absorbance reading at 280 nm indicate when measuring DNA purity?

    <p>Contamination by phenol or protein</p> Signup and view all the answers

    What is the absorbance range for accurate Spectrophotometer readings?

    <p>0.1–1.0</p> Signup and view all the answers

    What does the absorbance at 230 nm measure in a DNA sample?

    <p>Contamination by carbohydrates or salts</p> Signup and view all the answers

    What is a significant advantage of using Nanodrop Spectrophotometers over regular ones?

    <p>Higher sensitivity for low DNA concentrations</p> Signup and view all the answers

    What is the primary reason for using tracking dyes in gel electrophoresis?

    <p>To allow visual monitoring of electrophoresis progress</p> Signup and view all the answers

    Which of the following statements is true regarding DNA fragments in agarose gel?

    <p>DNA migration can be visually monitored using tracking dyes.</p> Signup and view all the answers

    What is a significant characteristic of ethidium bromide (EtBr)?

    <p>It intercalates between DNA bases and emits fluorescence upon UV exposure.</p> Signup and view all the answers

    What is the role of the anode in gel electrophoresis?

    <p>To attract negatively charged DNA fragments</p> Signup and view all the answers

    Why should ethidium bromide be handled with care in the laboratory?

    <p>It is a known mutagen and requires gloves during handling.</p> Signup and view all the answers

    What feature of RedSafe makes it preferable over ethidium bromide?

    <p>It is non-toxic and not mutagenic.</p> Signup and view all the answers

    What effect does DNA diffusion have on the observation of gel electrophoresis results?

    <p>It can blur the bands over time, making results less clear.</p> Signup and view all the answers

    What is the purpose of the UV transilluminator in visualizing DNA?

    <p>To visualize the stained DNA under UV light.</p> Signup and view all the answers

    What is the optimal concentration range of MgCl2 for standard PCR reactions?

    <p>1-4 mM</p> Signup and view all the answers

    How does excess Mg2+ affect DNA synthesis fidelity?

    <p>It stabilizes DNA double strands.</p> Signup and view all the answers

    What primer concentration range is typically recommended?

    <p>0.1 to 1 µM</p> Signup and view all the answers

    What is a consequence of high primer concentrations during PCR?

    <p>Increased likelihood of mis-priming</p> Signup and view all the answers

    Which condition is likely to improve the specificity of primer annealing?

    <p>Low primer concentration and high temperature</p> Signup and view all the answers

    What contributes to the formation of primer-dimers?

    <p>High primer concentration</p> Signup and view all the answers

    What is mis-priming in the context of DNA synthesis?

    <p>Primers annealing to a non-complementary sequence</p> Signup and view all the answers

    What is a key characteristic of a well-designed PCR primer?

    <p>It should be complementary to the coding strand.</p> Signup and view all the answers

    Study Notes

    Course Overview

    • Course: BIOD21 - Advanced Molecular Biology Lab
    • Instructor: Prof. Sonia Gazzarrini
    • Semester: Fall 2024

    Goal 1: Learning Lab Techniques

    • Planning and conducting experiments
      • Experimental plan (lab protocols/handouts)
      • Workflow
    • Data acquisition, interpretation, and troubleshooting
      • Careful description of each experimental step
      • Data discussion
      • Preparation of high-quality figures
    • Data presentation and discussion
      • Written lab report (research paper format)
      • Oral presentation (similar to lab meeting or conference presentations)

    Goal 2: Molecular Techniques for Gene Regulation

    • Transcriptional regulation in response to stress
      • RNA isolation from control and treatment (stress) plants
      • cDNA synthesis and RT-qPCR
    • Generation and characterization of mutants
      • Genome editing by CRISPR/Cas9
      • Phenotypic analysis of wild-type and mutants
    • Protein localization
      • Subcellular localization of proteins using a translational reporter (YFP fusion protein) and confocal microscopy
    • Gene cloning and sequencing
      • Cloning strategies (Gateway, Golden Gate)
      • Bacterial transformation, plasmid isolation, and colony screening by PCR
      • Sequencing data analysis

    Goal 2: Bioinformatics Analysis

    • Navigating molecular biology databases and repositories (NCBI, TAIR)
      • Using web-based tools for DNA and protein sequence analysis, alignment, primer design, etc.
      • In silico gene expression analysis using available transcriptomic data (microarray and RNAseq).
      • Primer and guide-RNA design
      • In-silico PCR
    • Alberts et al. (2002) Molecular Biology of the Cell (NCBI Bookshelf)
      • Chapter 8, sections on "Studying Gene Expression and Function" and "Isolating, Cloning, and Sequencing DNA" (NCBI Bookshelf links provided)
    • Lodish et al. (2000) Molecular Cell Biology (NCBI Bookshelf)
      • Section 7 "DNA cloning with plasmid vectors" (NCBI Bookshelf link provided)
    • Thermo Fisher Scientific (technical resources and reference library)
    • Addgene (protocols and in-house videos on Quercus)

    Overview of Weeks 1-5 Labs

    • Studying gene regulation (transcriptional regulation by abiotic stress) using the model organism Arabidopsis thaliana
    • In silico primer testing for qPCR
    • Using primers in genomic DNA using regular PCR (genomic DNA isolation and PCR)
    • Exposing WT plants to control and abiotic stress conditions and determining if CBF4 is transcriptionally regulated
    • In silico analyses of gene expression using available transcriptomic data (microarray and RNAseq) (Bioinformatics Lab 2)

    Week 1: Bioinformatics Lab 1

    • DNA sequence analysis and in silico PCR (NCBI)
      • CBF4 genomic, cDNA, and CDS sequences
    • Sequence alignment (Clustal Omega)
    • Primer design and in silico PCR (Primer Blast)
    • Bioinformatics tools for sequence analysis, including checking for introns, exons, and UTRs.

    Setting up PCR Reactions: Master Mix and Controls

    • Components of a PCR master mix
      • DNA
      • Enzyme (e.g., Taq polymerase)
      • dNTPs
      • Buffer (e.g., MgCl2)
      • Primers
    • Steps to prepare a master mix
      • Preparing a master mix with all components, and then adding each into a tube
    • Importance of negative and positive controls

    PCR Cycle

    • Steps in the PCR cycle:
      • Denaturation
      • Annealing
      • Extension
    • Optimizing conditions for different types of DNA

    Important Considerations

    • Primer specificity
      • Minimizing mis-priming: selecting primers to avoid regions of sequence similarity to other genes
      • Primer concentration
      • Primer length and GC content
      • Choosing primers with the correct Tm for annealing temperature
    • Primer-dimer and hairpin formation
    • Design of gene-specific primers

    Genomic DNA Isolation and Analysis

    • Procedure for genomic DNA isolation:
      • Sample crushing
      • Lysis buffer treatment
      • Supernatant transfer
      • Precipitation in isopropanol
      • Ethanol wash
      • Resuspension in TE buffer
    • Gel electrophoresis for genomic DNA analysis:
      • Agarose gel preparation and running
      • Staining and visualization of DNA fragments

    DNA and RNA Quantitation

    • Gel electrophoresis
    • Spectrophotometer
      • DNA concentration measurement using Nucleic acid intercalating dyes (uv illumination) and standards
    • Spectrophotometer-based quantitation of DNA
      • Use of absorbance at 260nm, 280nm, and 230nm
      • Calculating the ratio OD260/OD280
    • Limitations of methods

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    Related Documents

    Lecture 1-2 (DNA & PCR).pdf

    Description

    Test your knowledge on the Polymerase Chain Reaction (PCR) and gel electrophoresis in molecular biology. This quiz covers aspects such as the purpose of PCR, key enzymes involved, and the significance of agarose concentration in DNA fragment mobility. Perfect for students and enthusiasts looking to deepen their understanding of these essential techniques.

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