DNA Concentration Measurement Methods

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Questions and Answers

What method is directly proportional to the amount of DNA in a sample?

  • UV absorbance examination (correct)
  • DNA standard comparison
  • Fluorimetry
  • Agarose gel electrophoresis

What is the absorbance at 260 nm for a 50 µg/mL solution of double-stranded DNA?

  • 2.0
  • 1.0 (correct)
  • 0.5
  • 0.2

Which absorbance ratio indicates pure RNA?

  • 0.8
  • 2.0 (correct)
  • 1.8
  • 1.0

Which factor can lower the 260/280 absorbance ratios, resulting in reduced accuracy of DNA quantitation?

<p>Protein or phenol contamination (C)</p> Signup and view all the answers

What is the appropriate range of sample volume for accurate absorbance assessment in spectrophotometric measurement?

<p>5 to 50 µL (B)</p> Signup and view all the answers

What is the purpose of using a low percentage agarose gel during DNA concentration assessment?

<p>To separate nuclear and mitochondrial DNA (C)</p> Signup and view all the answers

What is the initial step before measuring a DNA sample's absorbance in a spectrophotometer?

<p>Warming up the UV-lamp (C)</p> Signup and view all the answers

What is the key requirement for preparing a test sample for spectrophotometric measurement?

<p>Uniform mixing of the sample (B)</p> Signup and view all the answers

What is the primary function of EDTA in the CTAB method?

<p>Chelate Mg 2+ to inactivate nucleases (D)</p> Signup and view all the answers

Which buffer is specifically used to separate plasmid DNA in gel electrophoresis?

<p>Alkaline buffer (A)</p> Signup and view all the answers

What role does chloroform isoamyl play in the CTAB method?

<p>Separates contaminants into organic phase (A)</p> Signup and view all the answers

How does the charge of molecules affect their migration during electrophoresis?

<p>Only negatively charged molecules move towards the positive electrode. (D)</p> Signup and view all the answers

Which of the following is NOT a component in the loading dye for gel electrophoresis?

<p>Isopropanol (B)</p> Signup and view all the answers

What is the purpose of using Ethidium bromide in gel electrophoresis?

<p>For staining DNA to visualize it (B)</p> Signup and view all the answers

What does the absorbance of RNA at 260 nm indicate in terms of concentration?

<p>0.04 μg/μL corresponds to 40 μg/mL (B)</p> Signup and view all the answers

In agarose gel electrophoresis, which property does the gel primarily separate molecules by?

<p>Charge, size, and shape (D)</p> Signup and view all the answers

What role does the loading dye play in preparing DNA samples for electrophoresis?

<p>It provides density so the samples can sink into the wells. (B)</p> Signup and view all the answers

Which of the following components is necessary for DNA detection after electrophoresis?

<p>Ethidium bromide (A)</p> Signup and view all the answers

What is the purpose of the electrophoresis buffer in the setup?

<p>It conducts electricity and controls pH. (D)</p> Signup and view all the answers

What happens to the smaller DNA molecules during electrophoresis in comparison to larger ones?

<p>They move through the pores of the gel more easily. (C)</p> Signup and view all the answers

How much agarose is recommended for preparing gel for a genomic DNA sample?

<p>0.8 g (A)</p> Signup and view all the answers

Which component in the 6X loading dye allows for tracking the migration progress?

<p>Bromophenol blue (B)</p> Signup and view all the answers

What is the primary charge of DNA molecules that dictates their movement towards the anode in electrophoresis?

<p>Negative charge (D)</p> Signup and view all the answers

What is the total volume of the tank buffer recommended for the electrophoresis cell?

<p>1500 mL (C)</p> Signup and view all the answers

What is the purpose of ethidium bromide in the agarose gel electrophoresis procedure?

<p>To visualize nucleic acids under UV light (A)</p> Signup and view all the answers

At which temperature should the agarose solution be cooled before pouring it into the gel tray?

<p>55°C (A)</p> Signup and view all the answers

What is the primary purpose of the STOP solution in DNA analysis?

<p>To halt enzymatic reactions (D)</p> Signup and view all the answers

Why is it necessary to wear protective eyewear when using a transilluminator?

<p>To prevent damage from ultraviolet light (B)</p> Signup and view all the answers

Which component is NOT part of the STBS solution?

<p>Sodium bicarbonate (C)</p> Signup and view all the answers

How does the pH of the TAE electrophoresis buffer differ from the pH of the STBS solution?

<p>STBS has a higher pH than TAE (C)</p> Signup and view all the answers

What is the concentration of ethidium bromide that is added to the agarose gel solution?

<p>0.5 µg/mL (B)</p> Signup and view all the answers

Which of the following accurately describes the components of the Storage buffer?

<p>Contains Tris-Cl and NaEDTA, pH 8 (A)</p> Signup and view all the answers

What happens to DNA fragments as their molecular weight increases when migrating through agarose gel?

<p>They migrate slower (B)</p> Signup and view all the answers

What is the effective concentration of EDTA in the STET solution?

<p>50 mM (C)</p> Signup and view all the answers

What is the correct method to prepare samples for loading into the agarose gel?

<p>Add 1 µL of gel loading dye for every 5 µL of DNA solution (A)</p> Signup and view all the answers

How long should the gel solidify at room temperature before use?

<p>20 minutes (D)</p> Signup and view all the answers

What should be done if the gel was not stained with ethidium during the run?

<p>The gel should be stained in 0.5 µg/mL ethidium bromide (B)</p> Signup and view all the answers

What is the primary purpose of using different agarose concentrations in gel electrophoresis?

<p>To enhance the resolution of different sizes of DNA fragments (D)</p> Signup and view all the answers

How does increasing voltage affect the migration of DNA fragments in gel electrophoresis?

<p>It allows larger fragments to migrate faster than smaller ones (D)</p> Signup and view all the answers

What is the consequence of using water instead of a buffer for DNA electrophoresis?

<p>DNA migration will effectively cease (B)</p> Signup and view all the answers

Which buffer is most commonly recommended for the electrophoresis of duplex DNA?

<p>Tris-borate-EDTA (TBE) (B)</p> Signup and view all the answers

What can be the effect of using a 10X concentrated buffer for gel electrophoresis?

<p>It may melt the gel due to excessive heat generation (D)</p> Signup and view all the answers

What is the primary role of agarose gel-loading buffer in DNA electrophoresis?

<p>To aid in the visibility and density of DNA samples for loading (C)</p> Signup and view all the answers

What is a correct final concentration of EDTA in the fusion protein extraction buffer?

<p>1% final (B)</p> Signup and view all the answers

Why are ionic strengths of buffers important in gel electrophoresis?

<p>They influence DNA migration rates (A)</p> Signup and view all the answers

Flashcards

DNA Concentration Measurement

Determining the amount of DNA in a sample, crucial for subsequent analyses.

Spectrophotometer (UV absorbance)

A method for measuring DNA concentration by quantifying UV light absorption by DNA in solution.

ds DNA Absorbance (260 nm)

50 µg/mL of dsDNA absorbs 1.0 at 260 nm.

ss DNA/RNA Absorbance (260 nm)

40 µg/mL of ssDNA or RNA absorbs 1.0 at 260 nm.

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260/280 Absorbance Ratio

Indicates DNA purity. Pure DNA has ratio ~1.8. Lower ratios suggest contaminants.

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Agarose Gel Electrophoresis

Used for assessing DNA quantity and quality (size, degradation), and RNA presence.

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Sample Volume (Spectrophotometry)

Volume of DNA sample needed for spectrophotometric analysis (5-50 µL).

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DNA Concentration (Typical values)

Genomic DNA ~100 µg/mL, plasmid DNA ~1 mg/mL (Approximate values).

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Agarose

A polysaccharide used to create a gel matrix in electrophoresis for molecule separation

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Electrophoresis

The movement of charged molecules in a gel matrix under the influence of an electric field.

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Loading Dye

Dye added to samples to improve visualization and density during electrophoresis using glycerol or sucrose

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DNA Separation by CTAB Method

A method including lysing cells, removing polysaccharides, proteins and separating DNA using specific buffers for DNA precipitation.

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RNA Absorbance

Measuring the amount of RNA in a solution, 40 µg/mL = 1.0 absorbance unit at 260 nm.

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CTAB Lysis Buffer

A buffer used to solubilize cells and complex with cellular nucleic acids, helping separate nucleic acid.

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Buffer (electrophoresis)

A solution having an almost constant pH, used in an electrophoresis apparatus to carry current and maintain the pH of separating solutions. Examples include TAE, TBE, TPE, or alkaline buffers.

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Electrophoresis Buffer

A solution that conducts electricity and controls pH, essential for DNA movement in electrophoresis.

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DNA Loading Dye

A solution mixed with DNA samples to make them denser and help them sink into the gel wells.

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Ethidium Bromide

A DNA-binding dye used to stain DNA fragments, making them visible under UV light.

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Agarose Gel Preparation - Genomic DNA

Process of making an agarose gel for separating genomic DNA, using 0.8g Agarose.

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Agarose Gel Preparation - PCR Product

Process of making an agarose gel for separating PCR product, using 1.5-2 g Agarose.

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Sample Wells

Indentation in the agarose gel where DNA samples are loaded.

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DNA Migration in Electrophoresis

Movement of negatively charged DNA fragments towards the positive electrode in an electric field.

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Transilluminator

An ultraviolet lightbox used to visualize DNA stained with ethidium bromide.

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Agarose Gel Preparation

Dissolving agarose powder in a buffer, allowing solidification in a tray to form a gel matrix for electrophoresis.

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DNA Fragment Size

Linear DNA fragments in an agarose gel migrate inversely proportional to the logarithm of their molecular weight or base pairs.

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DNA Gel Staining

Adding ethidium bromide to the gel or electrophoresis buffer after electrophoresis to visualize the DNA bands.

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STE buffer

A buffer commonly used for extracting DNA, containing Tris-Cl, NaCl, and EDTA.

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STET solution

A solution containing sucrose, Triton X-100, EDTA, and Tris-Cl, used for lysis and cell disruption.

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Suspension Medium (SM)

A solution used to suspend and store bacteria, containing NaCl, MgSO4, Tris-Cl, gelatin, and water.

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T4 DNA ligase Buffer

A buffer used for ligating DNA fragments, containing Tris-Cl, MgCl2, and DTT.

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TA buffer

A buffer used for cloning and transformation reactions, containing Tris-acetate, potassium acetate, magnesium acetate, DTT, and BSA.

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Agarose Concentration

The percentage of agarose in a gel determines which size DNA fragments are best separated.

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Voltage Effect on Gel

Increasing voltage makes larger DNA fragments migrate faster, impacting separation.

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Why do we use buffers?

Buffers provide ions for conductivity and maintain a stable pH during electrophoresis.

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Buffer Importance

Using a buffer is critical for DNA migration in gels. Without it, DNA won't move.

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Concentrated Buffer Effects

Using a concentrated buffer can generate excessive heat, potentially melting the gel.

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Gel-Loading Buffer

A solution containing tracking dyes and sucrose, added to DNA samples before loading onto a gel.

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Study Notes

DNA Concentration Measurement

  • Determining DNA concentration is crucial before further analysis
  • Three methods exist: UV absorbance, fluorimetry, and comparison with DNA standards
  • UV spectrophotometry measures the UV radiation absorbed by DNA solution
  • Absorbance is directly proportional to DNA concentration
  • Double-stranded DNA (dsDNA) at 50 µg/ml has an absorbance of 1.0 at 260 nm
  • Single-stranded DNA (ssDNA) and RNA at 40 µg/ml have an absorbance of 1.0 at 260 nm
  • Purity assessment relies on the 260/280 nm absorbance ratio
  • A ratio of 1.8 for DNA and 2.0 for RNA indicates purity
  • Protein or phenol contamination reduces the accuracy and lowers ratios
  • RNA and pigments in DNA preparations lead to inaccurate estimations
  • 0.8% agarose gels help assess DNA quantity and quality (size, shearing)
  • 0.4% agarose gels help distinguish between nuclear and mitochondrial DNA

Spectrophotometric DNA Concentration Determination

  • Turn on UV lamp, allow warming up, and set wavelength to 260nm
  • Prepare test samples by adding TE to 500µl final volume and mix
  • Fill one cuvette with 500µl TE and measure absorbance at 260nm (Zero)
  • Fill another cuvette with 500µl sample & measure at both 260nm & 280nm
  • The blank (TE) and sample readings are noted
  • Rinse cuvettes and repeat step 6 for each sample
  • Rinse and dry cuvette to repeat step 6 for each sample.
  • Calculate DNA concentration considering initial dilution

DNA Concentration Calculation

  • DNA concentration (µg/ml) = (500 µl / x µl) * OD260
  • x is the volume of DNA used

Agarose Gel Electrophoresis

  • Separates molecules (DNA, RNA, proteins) based on size and charge
  • Agarose is a polysacharide that creates a molecular sieve
  • Smaller molecules migrate faster than larger ones
  • DNA samples are mixed with glycerol or sucrose to increase density
  • Samples are loaded into wells and subjected to electric field
  • Charged molecules move to the oppositely charged electrode (anode)
  • Agarose concentration affects fragment separation (higher concentration for smaller fragments)
  • Voltage and buffer affect migration rates
  • Buffer ensures appropriate pH for conductivity and molecule stability.

DNA Staining

  • Ethidium bromide is a fluorescent dye to visualize DNA fragments

Buffers for Gel Electrophoresis

  • TAE (Tris-acetate-EDTA), and TBE (Tris-borate-EDTA) buffers are used
  • Various buffer solutions like STE, AP buffer, Citrate/Saline buffer have role in DNA analysis and separation under different conditions.

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