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Questions and Answers
In spectrophotometry, what property of a substance is measured to determine its concentration?
In spectrophotometry, what property of a substance is measured to determine its concentration?
- Light emission
- Electrical charge
- Light absorption (correct)
- Particle size
Which of the following factors affects the rate at which biomolecules move through a gel matrix during electrophoresis?
Which of the following factors affects the rate at which biomolecules move through a gel matrix during electrophoresis?
- The charge, size, and shape of the biomolecules (correct)
- The color of the biomolecules
- The temperature of the laboratory
- The researcher's preference
- The type of buffer used in the gel
According to the Beer-Lambert Law, what is the relationship between absorbance and concentration of a substance?
According to the Beer-Lambert Law, what is the relationship between absorbance and concentration of a substance?
- Absorbance is inversely proportional to the concentration.
- Absorbance is independent of the concentration.
- Absorbance is directly proportional to the concentration. (correct)
- Absorbance is logarithmically related to the concentration.
What wavelength is typically used to measure the absorbance of nucleic acids in spectrophotometry?
What wavelength is typically used to measure the absorbance of nucleic acids in spectrophotometry?
If a DNA sample diluted 1/100 gives an absorbance reading of 0.300 at 260 nm, what is the concentration of the DNA in μg/mL, assuming a conversion factor of 50 μg/mL per absorbance unit?
If a DNA sample diluted 1/100 gives an absorbance reading of 0.300 at 260 nm, what is the concentration of the DNA in μg/mL, assuming a conversion factor of 50 μg/mL per absorbance unit?
What is the purpose of using fluorescent dyes like ethidium bromide or SybrGreen in electrophoresis?
What is the purpose of using fluorescent dyes like ethidium bromide or SybrGreen in electrophoresis?
What does an A260/A280 ratio of approximately 1.8 indicate about a DNA sample?
What does an A260/A280 ratio of approximately 1.8 indicate about a DNA sample?
Which of the following methods is commonly used to analyze very small amounts of fluid in microfluidic systems?
Which of the following methods is commonly used to analyze very small amounts of fluid in microfluidic systems?
What is the purpose of the quartz cuvette in UV spectrometry?
What is the purpose of the quartz cuvette in UV spectrometry?
Which of the following best describes the function of NanoDrop spectrophotometry?
Which of the following best describes the function of NanoDrop spectrophotometry?
In fluorometry, what is measured to determine DNA concentration?
In fluorometry, what is measured to determine DNA concentration?
Which of the following is a modern dye used in fluorometry that binds to adenine-thymine base pairs in the minor groove of DNA?
Which of the following is a modern dye used in fluorometry that binds to adenine-thymine base pairs in the minor groove of DNA?
How does the principle of electrophoresis aid in analyzing nucleic acids?
How does the principle of electrophoresis aid in analyzing nucleic acids?
What might an A260/A280 ratio lower than 1.6 indicate about a nucleic acid sample?
What might an A260/A280 ratio lower than 1.6 indicate about a nucleic acid sample?
What is a key advantage of using microfluidics in nucleic acid analysis?
What is a key advantage of using microfluidics in nucleic acid analysis?
Why might absorption and fluorometry readings sometimes disagree when quantifying nucleic acids?
Why might absorption and fluorometry readings sometimes disagree when quantifying nucleic acids?
A researcher needs to quantify a very dilute DNA sample (15 ng/mL). Which method would be most suitable?
A researcher needs to quantify a very dilute DNA sample (15 ng/mL). Which method would be most suitable?
What is indicated by the RNA Integrity Number (RIN) in microfluidic analysis?
What is indicated by the RNA Integrity Number (RIN) in microfluidic analysis?
Which of the following is a primary advantage of using spectrophotometry over other methods for nucleic acid quantification?
Which of the following is a primary advantage of using spectrophotometry over other methods for nucleic acid quantification?
How does NanoDrop spectrophotometry improve upon standard UV spectrophotometry?
How does NanoDrop spectrophotometry improve upon standard UV spectrophotometry?
What information does electrophoresis provide about a DNA or RNA sample?
What information does electrophoresis provide about a DNA or RNA sample?
What is the correct formula for Beer-Lambert Law?
What is the correct formula for Beer-Lambert Law?
If given the values $A = 0.5$, $b = 1 cm$, and $e = 25 L/(mol \cdot cm)$, what is the concentration $c$ according to Beer-Lambert Law?
If given the values $A = 0.5$, $b = 1 cm$, and $e = 25 L/(mol \cdot cm)$, what is the concentration $c$ according to Beer-Lambert Law?
What unit is used to measure molar absorptivity ($e$) in the Beer-Lambert Law?
What unit is used to measure molar absorptivity ($e$) in the Beer-Lambert Law?
What is the path length $b$ in Beer-Lambert Law?
What is the path length $b$ in Beer-Lambert Law?
In microfluidics, what type of sample is typically applied to a multi-well chip for analysis?
In microfluidics, what type of sample is typically applied to a multi-well chip for analysis?
What type of output does instrument software typically generate in microfluidics?
What type of output does instrument software typically generate in microfluidics?
Why is microfluidics particularly useful for studying small RNAs (microRNAs) in eukaryotes and gene expression in bacteria?
Why is microfluidics particularly useful for studying small RNAs (microRNAs) in eukaryotes and gene expression in bacteria?
Which of the following is NOT a characteristic of UV Spectrometry?
Which of the following is NOT a characteristic of UV Spectrometry?
What does fluorometry measure to determine DNA concentration?
What does fluorometry measure to determine DNA concentration?
Which modern dye combines adenine-thymine base pairs in the minor groove of the DNA double helix??
Which modern dye combines adenine-thymine base pairs in the minor groove of the DNA double helix??
OliGreen can detect down to _____ of ssDNA?
OliGreen can detect down to _____ of ssDNA?
What does indication of contamination mean?
What does indication of contamination mean?
Given these values are correct, which sample is suitable for use of DNA?
Sample 1: 0.419/0.230 = 1.8
Sample 2: 0.258/0.225 = 1.1
Sample 3: 0.398/0.174 = 2.2
Given these values are correct, which sample is suitable for use of DNA? Sample 1: 0.419/0.230 = 1.8 Sample 2: 0.258/0.225 = 1.1 Sample 3: 0.398/0.174 = 2.2
Which measurement is most associated with fluorometry?
Which measurement is most associated with fluorometry?
Which of the following techniques would be the MOST appropriate for separating charged biomolecules based on size?
Which of the following techniques would be the MOST appropriate for separating charged biomolecules based on size?
What is the function of an electric current during gel electrophoresis?
What is the function of an electric current during gel electrophoresis?
When quantifying a nucleic acid sample, a researcher detects an unusually high absorbance at 230 nm. What does this suggest about the sample?
When quantifying a nucleic acid sample, a researcher detects an unusually high absorbance at 230 nm. What does this suggest about the sample?
Flashcards
Electrophoresis
Electrophoresis
Separates particles using an electric current through a solution or matrix.
Spectrophotometry
Spectrophotometry
Technique measuring a substance's concentration by how much light it absorbs.
Fluorometry
Fluorometry
Measurement of light emitted after a substance absorbs light.
Microfluidics
Microfluidics
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Electrophoresis for Nucleic Acids
Electrophoresis for Nucleic Acids
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260 nm Absorption
260 nm Absorption
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Beer-Lambert Law
Beer-Lambert Law
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A260/A280 ratio for DNA
A260/A280 ratio for DNA
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A260/A280 ratio for RNA
A260/A280 ratio for RNA
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UV Spectrometry
UV Spectrometry
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Fluorometry (Fluorescent Spectroscopy)
Fluorometry (Fluorescent Spectroscopy)
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Absorption vs Fluorometry
Absorption vs Fluorometry
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NanoDrop Spectrophotometry
NanoDrop Spectrophotometry
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RNA Integrity Number (RIN)
RNA Integrity Number (RIN)
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Study Notes
- Measurement of nucleic acid quality and quantity are essential in molecular biology.
Electrophoresis
- A separation technique, separates particles through a medium under an electric field.
- Used to assess the quality of DNA and RNA by separating an aliquot of the sample on agarose gel.
- An electric current propels charged biomolecules through a porous gel matrix.
- The rate of movement depends on the charge, size, and shape of the molecules.
- Fluorescent dyes such as ethidium bromide, SybrGreen I & II, and silver stain help visualize DNA.
- DNA appearance on agarose gels depends on the type of DNA isolated.
Spectrophotometry
- A technique that measures the concentration of an analyte by measuring light absorption.
- Nucleic acids absorb light at 260 nm through the adenine residues.
- Beer-Lambert Law: A = εbc (A = absorbance, ε = molar absorptivity, b = path length, c = concentration).
- Absorbance is directly proportional to the nucleic acid concentration in the sample.
- Example: DNA diluted 1/100 with a 0.200 absorbance at 260 nm has a concentration of 1,000 ug/mL.
- To calculate yield, multiply concentration by volume (e.g., 1,000 ug/mL x 0.5 mL = 500 ug).
- Molar absorptivity (ε) is 50 L/mol-cm for dsDNA and 40 for RNA.
- At 260 nm, 1 optical density unit = 50 mg/L (dsDNA) or 40 pg/mL (RNA).
- Concentration calculation: spectrophotometer reading x conversion factor (x dilution factor if needed).
Fluorometry
- Measures the emitted fluorescent light from a sample.
- Fluorescent light emitted is associated with DNA-specific fluorescent dyes.
- Used to detect small amounts of analytes.
Microfluidics
- Uses systems that process or manipulate small fluid volumes via microchannels.
- Uses capillaries.
Purity Estimation
- Contamination is indicated by absorbance over background outside the A260 maxima.
- A260/A280 ratio assesses DNA purity, reference values for DNA and RNA are needed.
UV Spectrometry
- Standard nucleic acid quantitation method.
- UV light is passed through a sample in a quartz cuvette and absorbance is measured at specific wavelengths.
- No additional reagents or incubation time is required.
Fluorometry (Fluorescent Spectroscopy)
- Relates fluorescence to DNA concentration using DNA-specific fluorescent dyes.
- Improved methods such as PicoGreen and OliGreen are utilized.
Agreements
- Absorption measurements may not distinguish between DNA and RNA.
NanoDrop Spectrophotometry
- Similar to standard spectrophotometry, but with additional features.
- Combines fiber optic technology and natural surface tension.
- Special software analyzes signals from small sample quantities.
- Displays the entire absorbance spectrum and detects contaminants.
Microfluidics
- Sample moves through microchannels on a multi-well chip to a detector.
- Instruments generate electropherogram or gel images.
- RNA integrity number quantifies RNA integrity.
- Requires minimal sample volume and can test multiple samples simultaneously.
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