CB Lab 8

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Questions and Answers

What is the primary function of agarose gel electrophoresis?

To measure DNA concentration

To identify DNA sequences

To separate DNA fragments by size (correct)

To amplify DNA samples

Which factor affects the migration speed of DNA in agarose gel?

Gel concentration (correct)

Volume of the buffer solution

Temperature of the agarose

Color of the DNA sample

In which direction does DNA migrate during electrophoresis?

Towards the positive electrode (correct)

Randomly within the gel

Towards the negative electrode

Not at all

What is the relationship between DNA size and migration through an agarose gel?

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

What is the effect of increasing the applied voltage during electrophoresis?

Faster migration of DNA fragments Signup and view all the answers

Which statement about DNA conformation and its effect on migration is correct?

Compact DNA shapes migrate faster than linear forms of the same size Signup and view all the answers

What happens to the resolution of DNA separation as the agarose concentration increases?

Resolution increases due to less pore size Signup and view all the answers

Which of the following describes gel electrophoresis compared to a method like SDS-PAGE?

Agarose gel has lower resolving power than SDS-PAGE Signup and view all the answers

What is the purpose of mixing the sample with loading dye in agarose gel electrophoresis?

To provide density to the sample Signup and view all the answers

Which component is NOT mentioned as part of the agarose gel electrophoresis procedure?

Ethanol Signup and view all the answers

What structural feature differentiates RT-PCR products from PCR products when analyzing At-ACTIN?

Absence of introns in cDNA Signup and view all the answers

What is the purpose of visualizing agarose gel under UV light?

To detect DNA bands stained with SYBR gold Signup and view all the answers

During PCR, what primarily determines the annealing temperature?

Specificity of the primers Signup and view all the answers

What concentration of agarose is used to prepare the gel according to the procedure?

1% Signup and view all the answers

Agarose Gel Electrophoresis

The gel is casted horizontally Separates large molecules based on size Commonly used for DNA separations DNA moves from the negative (black) electrode to the positive (red) electrode Lower resolving power than SDS-PAGE Signup and view all the answers

Agarose concentration

Agarose concentration (0.5% - 2%) <ul> <li>The mobility of DNA molecules is inversely proportional to gel concentration.</li> </ul> Signup and view all the answers

DNA conformation

DNA conformation <ul> <li>DNA molecules having a more compact shape (e.g. plasmid DNA) moves faster through the gel compared with linear DNA fragments of the same size.</li> </ul> Signup and view all the answers

d. Applied voltage

d. Applied voltage (50 V – 150 V) <ul> <li>Within a range, the higher the applied voltage, the faster the sample migration.</li> </ul> Signup and view all the answers

gel stains with what and look at under UV

SYBR gold Signup and view all the answers

Study Notes

Lab 8: Nucleic Acids Separation by Agarose Gel Electrophoresis

Lab course: BIOL 3120 Cell Biology
Date: 10/28/2024
Lecturer: Rubaia Tasmin, Master's Student

Today's Lab Overview

Agarose Gel Electrophoresis:
- Principle: Separates DNA fragments based on size in an agarose gel. Higher agarose concentration = smaller pore size.
- DNA migration: DNA moves from the negative (black) electrode to the positive (red) electrode. Smaller fragments move faster through the gel.
- Factors affecting DNA migration:
  - Agarose concentration: Inversely proportional to DNA mobility.
  - Size of DNA molecule: Smaller molecules move faster.
  - DNA conformation: Compact molecules (like plasmid DNA) move faster than linear fragments of the same size
  - Applied voltage: Higher voltage = faster migration (within a 50-130 Volt range)

Procedures of Agarose Gel Electrophoresis

Agarose gel preparation: 1g of agarose in 100mL TAE buffer.
Sample preparation: Samples mixed with loading dye (bromophenol blue and sucrose for density).
Gel polymerization: Agarose gel solidifies as it cools.
Loading samples into wells: DNA samples added into wells in gel.
Visualization under UV light: Gel visualized using SYBR gold stain.

Post-lab Questions

RT-PCR Product size vs. Genomic PCR Product: Different sizes due to differences in the starting materials and potential intron/exon differences in the template sequence.
PCR Annealing Temperature: Depends on primer sequence and the melting temperature of the primer pairs.
PCR extension time: Determined by the length of DNA sequence targeted for the amplification, reaction components, and the buffer.
Primers for sequence amplification: Sequence provided for primer design (page 11).

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Description

Explore the principles and procedures of Agarose Gel Electrophoresis in BIOL 3120 Cell Biology. This lab focuses on the separation of DNA fragments based on size and the factors influencing DNA migration. Enhance your understanding of this crucial technique with this informative quiz.