Fluorescence Lifetime Measurements and Pulsed Excitation

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What is the purpose of selecting excitation and emission wavelengths in fluorescence microscopy?

To control the direction of fluorescence

To control the color of fluorescence

To control the intensity of fluorescence (correct)

To control the duration of fluorescence

Which excitation light source is commonly used in fluorescence microscopes due to its lower energy consumption and longer lifetime compared to other light sources?

Mercury vapor lamp

Metal halide lamp

High power LED (correct)

Xenon arc lamp

What type of semiconductor is formed by doping silicon with a trivalent element like indium or boron?

Intrinsic semiconductor

N-type semiconductor

Extrinsic semiconductor

P-type semiconductor (correct)

What happens at a p-n junction in a diode?

All of the above Signup and view all the answers

Which of the following statements about bias in a p-n junction is correct?

Reverse bias increases the width of the depletion zone. Signup and view all the answers

What happens when a photon of sufficient energy strikes a photodiode?

An electron-hole pair is created in the depletion region. Signup and view all the answers

What is the purpose of the dynodes in a photomultiplier tube (PMT)?

To amplify the current produced by incident light. Signup and view all the answers

What is the quantum yield of a molecule?

The ratio of emitted photons to absorbed photons. Signup and view all the answers

Which of the following is true about light absorption?

Light absorption decreases the intensity of transmitted light. Signup and view all the answers

What is the speed of light in a vacuum?

3 x 10^8 m/s Signup and view all the answers

Which of the following interactions of light with matter can reveal information about the sample or properties of its surface?

Absorption and re-emission Signup and view all the answers

Which wavelengths are typically used in optical methods for bioanalysis?

Visible and ultraviolet wavelengths Signup and view all the answers

Which of the following is true about absorption spectrum?

Absorption spectrum is a scan over wavelengths. Signup and view all the answers

What is the ratio A260/A280 used for?

To determine the protein contamination in a DNA solution. Signup and view all the answers

What does the Beer-Lambert law relate?

The attenuation of light to the properties of the material. Signup and view all the answers

What is the purpose of a NanoDrop instrument?

To measure the concentration of tiny volumes of sample. Signup and view all the answers

Which process involves the release of energy absorbed by a substance in the form of light?

Phosphorescence Signup and view all the answers

What is the main difference between fluorescent materials and phosphorescent materials?

Fluorescent materials emit light immediately when the radiation source stops, while phosphorescent materials continue to emit light for some time after Signup and view all the answers

What is a fluorochrome?

A label used in biological research that fluoresces Signup and view all the answers

Why does the emitted photon usually have less energy than the absorbed photon in fluorescence?

Some of the energy is dissipated as heat Signup and view all the answers

Which type of quenching occurs when the fluorophore is in the excited state and collisions with other species result in relaxation of the fluorophore without photon emission?

Dynamic quenching Signup and view all the answers

What is the efficiency of energy transfer (E) in fluorescence resonance energy transfer (FRET) experiments?

The fraction of photon energy that is absorbed by the donor and transferred to the acceptor. Signup and view all the answers

What is the rate of energy transfer (ket) in the Förster equation for fluorescence resonance energy transfer (FRET)?

The fluorescence lifetime of the donor in the absence of the acceptor molecule. Signup and view all the answers

What is the process of transferring energy from a fluorophore in the excited state to a fluorescent or non-fluorescent acceptor molecule called?

Fluorescence resonance energy transfer (FRET) Signup and view all the answers

Which of the following is a major application of fluorescence spectroscopy in biology?

Tracking tagged molecules in complex environments Signup and view all the answers

What is a limitation of conventional fluorescence microscopes?

They cannot image thick specimens like tissue samples Signup and view all the answers

Which of the following is a mutant of GFP that is fluorescent in a different wavelength region?

Yellow fluorescent protein (YFP) Signup and view all the answers

What can fluorescence microscopy be used to observe in cells?

The presence of target molecules in distinct areas Signup and view all the answers

Which type of microscope is commonly used to obtain images of individual cells?

Bright-field microscope Signup and view all the answers

In what type of culture are mammalian cells typically grown in the lab?

Adherent two-dimensional culture Signup and view all the answers

Which scientist first coined the term 'plasmid' in 1952?

Joshua Lederberg Signup and view all the answers

What is a plasmid?

A small DNA molecule within a cell that can replicate independently Signup and view all the answers

What is the normal number of copies of plasmid found in a single cell called?

Copy number Signup and view all the answers

What is the purpose of CsCl density gradient centrifugation in isolating plasmids?

To separate chromosomal DNA from plasmid DNA Signup and view all the answers

Which of the following is a key factor that limits the number of DNA molecules that can be achieved in a PCR reaction?

The depletion of primers and nucleotides Signup and view all the answers

What is the purpose of real-time PCR?

To determine the amount of starting template in different samples Signup and view all the answers

Which detection scheme is commonly used in PCR reactions to track the progress of the reaction?

Intercalating dye Signup and view all the answers

What is the efficiency of a PCR reaction represented by?

x Signup and view all the answers

Which enzyme is added to the pyrosequencing reaction mixture to catalyze the reaction of pyrophosphate (PPi) with adenosine 5’ phosphosulfate (APS)?

ATP sulfurylase Signup and view all the answers

What triggers the release of visible light in pyrosequencing?

Luciferase Signup and view all the answers

What is the limitation of pyrosequencing when it comes to detecting homopolymeric regions?

Homopolymeric regions longer than 10 bases cannot be resolved Signup and view all the answers

What is the purpose of using deoxyadenosine α-thio triphosphate (dATP-αS) instead of natural dATP in pyrosequencing?

To interfere with the luciferase reaction Signup and view all the answers

Which of the following is true about Ct values in real-time PCR?

Ct values indicate the number of cycles it took to detect a real signal above background fluorescence Signup and view all the answers

What is the formula used to calculate fold changes in gene expression in qRT-PCR?

Fold change = 2^-Δ(ΔCt) Signup and view all the answers

What is the purpose of relative quantification in gene expression analysis?

To normalize Ct values to a housekeeping gene and a baseline experimental condition Signup and view all the answers

What is a disadvantage of qPCR compared to other methods?

Expensive equipment and technical expertise required Signup and view all the answers

Which detection method in real-time PCR involves the use of two sequence-specific oligonucleotide probes labeled with different dyes?

Detection Method 3 Signup and view all the answers

What is the purpose of melting curve analysis in real-time PCR?

To assess the dissociation characteristics of double-stranded DNA Signup and view all the answers

What is reverse transcription?

The synthesis of single-stranded DNA using single-stranded RNA as a template Signup and view all the answers

Which type of virus uses RNA as its genetic material and replicates through reverse transcription?

Retrovirus Signup and view all the answers

Which of the following methods can be used to isolate DNA and RNA from living cells?

Treatment with an enzyme that has cell lytic properties Signup and view all the answers

What is the purpose of centrifuging a sample mixed with cesium chloride (CsCl) at very high speeds in an ultracentrifuge?

To separate DNA from RNA and protein based on their buoyant density Signup and view all the answers

Which of the following is true about the separation of DNA, RNA, and protein using CsCl centrifugation?

Proteins have the lowest density and float on the top of the tube Signup and view all the answers

How can the separated DNA be removed from the centrifugation tube?

By using a syringe Signup and view all the answers

Which of the following is a common method used to select specifically for mRNAs in RNA-seq experiments?

Polyadenylation Signup and view all the answers

What is the purpose of second strand synthesis in RNA-seq experiments?

To capture an entire transcript Signup and view all the answers

What is the role of ribonuclease H (RNase H) in RNA-seq experiments?

To cleave RNA in RNA:DNA hybrids Signup and view all the answers

Why might random hexamers be used instead of oligo(dT) primers for first strand cDNA synthesis in RNA-seq experiments?

To avoid 3' end bias in the sequencing library Signup and view all the answers

What is the purpose of adapter ligation in RNA-seq library construction?

To enable sequencing of the cDNA fragments Signup and view all the answers

Why are ribosomal RNAs (rRNAs) often depleted from the total RNA pool in RNA-seq experiments?

Because they are highly abundant and can dominate sequencing reads Signup and view all the answers

Which enzyme is added to the pyrosequencing reaction mixture to catalyze the reaction of pyrophosphate (PPi) with adenosine 5’ phosphosulfate (APS)?

ATP sulfurylase Signup and view all the answers

What triggers the release of visible light in pyrosequencing?

Oxyluciferin Signup and view all the answers

What is the current market share of the Asian market in the global NGS market as of 2017?

22.5% Signup and view all the answers

Which sequencing technology uses reversible terminators?

Illumina sequencing Signup and view all the answers

Which of the following statements about plasmids is true?

Plasmids are large circular DNA molecules found in bacteria. Signup and view all the answers

What is the purpose of CsCl density gradient centrifugation in isolating plasmids?

To separate chromosomal DNA from plasmid DNA based on their density. Signup and view all the answers

What is the purpose of phenol-chloroform extraction in DNA or RNA purification?

To remove any contaminating phenol from the DNA or RNA solution. Signup and view all the answers

What is the purpose of minipreparation of plasmid DNA?

To isolate plasmid DNA from bacteria on a large scale. Signup and view all the answers

Which detection method in real-time PCR involves the use of a dye that intercalates with double stranded DNA and causes fluorescence?

Detection Method 1 Signup and view all the answers

Which detection method in real-time PCR involves the use of a probe that is cleaved by the polymerase enzyme, resulting in increased fluorescence?

Detection Method 2 Signup and view all the answers

Which detection method in real-time PCR involves the use of two sequence-specific oligonucleotide probes labeled with different dyes?

Detection Method 3 Signup and view all the answers

What is the purpose of melting curve analysis in real-time PCR?

To assess the dissociation characteristics of double-stranded DNA during heating Signup and view all the answers

Which of the following is a key factor that limits the number of DNA molecules that can be achieved in a PCR reaction?

The efficiency of the reaction Signup and view all the answers

What is the formula used to calculate the theoretically predicted number of DNA molecules, Nm, at the end of a PCR reaction?

Nm = N0 * (2^n) Signup and view all the answers

What is the purpose of real-time PCR?

To monitor the formation of products as the reaction proceeds Signup and view all the answers

What are the two major detection schemes commonly used to track the progress of a PCR reaction?

Intercalating dye and hydrolysis probe Signup and view all the answers

Which of the following is a method used to isolate DNA and RNA from living cells?

Treatment with an enzyme that has cell lytic properties Signup and view all the answers

What is the purpose of CsCl density gradient centrifugation in isolating plasmids?

To concentrate DNA molecules close to the middle of the tube Signup and view all the answers

What triggers the release of visible light in pyrosequencing?

Hydrolysis of pyrophosphate Signup and view all the answers

What is the purpose of adapter ligation in RNA-seq library construction?

To add sequencing adapters to the RNA fragments Signup and view all the answers

Which of the following is true about Ct values in real-time PCR?

Ct values are inversely proportional to the amount of targeted nucleic acid in the sample. Signup and view all the answers

What is the purpose of a standard curve in absolute quantification of gene expression?

To compare results from actual experiments with known amounts of target DNA. Signup and view all the answers

What is the purpose of relative quantification in gene expression analysis?

To calculate fold changes in gene expression. Signup and view all the answers

What is the purpose of the cleavable hairpin beacon (CHB) in colorimetric LAMP?

To increase the target specificity of colorimetric LAMP. Signup and view all the answers

Which of the following is a key application of RNA-seq?

All of the above Signup and view all the answers

What is the purpose of depleting ribosomal RNAs (rRNAs) in RNA-seq experiments?

To remove highly abundant rRNAs from the total RNA pool Signup and view all the answers

What is the main advantage of using oligo(dT) beads for RNA-seq?

They specifically capture mRNAs Signup and view all the answers

What is the purpose of using random hexamers for first strand cDNA synthesis in RNA-seq?

To capture an entire transcript without 3' end bias Signup and view all the answers

What is the purpose of adding an 'A' base to the 3' ends of dsDNAs in RNA-seq library construction?

To facilitate adapter ligation Signup and view all the answers

What is the purpose of Y-shaped adapters in Illumina library preparation for RNA-seq?

To facilitate adapter ligation Signup and view all the answers

Fluorescence Lifetime Measurements and Pulsed Excitation

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Questions and Answers

What is the purpose of selecting excitation and emission wavelengths in fluorescence microscopy?

Which excitation light source is commonly used in fluorescence microscopes due to its lower energy consumption and longer lifetime compared to other light sources?

What type of semiconductor is formed by doping silicon with a trivalent element like indium or boron?

What happens at a p-n junction in a diode?

Which of the following statements about bias in a p-n junction is correct?

What happens when a photon of sufficient energy strikes a photodiode?

What is the purpose of the dynodes in a photomultiplier tube (PMT)?

What is the quantum yield of a molecule?

Which of the following is true about light absorption?

What is the speed of light in a vacuum?

Which of the following interactions of light with matter can reveal information about the sample or properties of its surface?

Which wavelengths are typically used in optical methods for bioanalysis?

Which of the following is true about absorption spectrum?

What is the ratio A260/A280 used for?

What does the Beer-Lambert law relate?

What is the purpose of a NanoDrop instrument?

Which process involves the release of energy absorbed by a substance in the form of light?

What is the main difference between fluorescent materials and phosphorescent materials?

What is a fluorochrome?

Why does the emitted photon usually have less energy than the absorbed photon in fluorescence?

Which type of quenching occurs when the fluorophore is in the excited state and collisions with other species result in relaxation of the fluorophore without photon emission?

What is the efficiency of energy transfer (E) in fluorescence resonance energy transfer (FRET) experiments?

What is the rate of energy transfer (ket) in the Förster equation for fluorescence resonance energy transfer (FRET)?

What is the process of transferring energy from a fluorophore in the excited state to a fluorescent or non-fluorescent acceptor molecule called?

Which of the following is a major application of fluorescence spectroscopy in biology?

What is a limitation of conventional fluorescence microscopes?

Which of the following is a mutant of GFP that is fluorescent in a different wavelength region?

What can fluorescence microscopy be used to observe in cells?

Which type of microscope is commonly used to obtain images of individual cells?

In what type of culture are mammalian cells typically grown in the lab?

Which scientist first coined the term 'plasmid' in 1952?

What is a plasmid?

What is the normal number of copies of plasmid found in a single cell called?

What is the purpose of CsCl density gradient centrifugation in isolating plasmids?

Which of the following is a key factor that limits the number of DNA molecules that can be achieved in a PCR reaction?

What is the purpose of real-time PCR?

Which detection scheme is commonly used in PCR reactions to track the progress of the reaction?

What is the efficiency of a PCR reaction represented by?

Which enzyme is added to the pyrosequencing reaction mixture to catalyze the reaction of pyrophosphate (PPi) with adenosine 5’ phosphosulfate (APS)?

What triggers the release of visible light in pyrosequencing?

What is the limitation of pyrosequencing when it comes to detecting homopolymeric regions?

What is the purpose of using deoxyadenosine α-thio triphosphate (dATP-αS) instead of natural dATP in pyrosequencing?

Which of the following is true about Ct values in real-time PCR?

What is the formula used to calculate fold changes in gene expression in qRT-PCR?

What is the purpose of relative quantification in gene expression analysis?

What is a disadvantage of qPCR compared to other methods?

Which detection method in real-time PCR involves the use of two sequence-specific oligonucleotide probes labeled with different dyes?

What is the purpose of melting curve analysis in real-time PCR?

What is reverse transcription?

Which type of virus uses RNA as its genetic material and replicates through reverse transcription?

Which of the following methods can be used to isolate DNA and RNA from living cells?

What is the purpose of centrifuging a sample mixed with cesium chloride (CsCl) at very high speeds in an ultracentrifuge?

Which of the following is true about the separation of DNA, RNA, and protein using CsCl centrifugation?

How can the separated DNA be removed from the centrifugation tube?

Which of the following is a common method used to select specifically for mRNAs in RNA-seq experiments?

What is the purpose of second strand synthesis in RNA-seq experiments?

What is the role of ribonuclease H (RNase H) in RNA-seq experiments?

Why might random hexamers be used instead of oligo(dT) primers for first strand cDNA synthesis in RNA-seq experiments?

What is the purpose of adapter ligation in RNA-seq library construction?

Why are ribosomal RNAs (rRNAs) often depleted from the total RNA pool in RNA-seq experiments?

Which enzyme is added to the pyrosequencing reaction mixture to catalyze the reaction of pyrophosphate (PPi) with adenosine 5’ phosphosulfate (APS)?

What triggers the release of visible light in pyrosequencing?

What is the current market share of the Asian market in the global NGS market as of 2017?

Which sequencing technology uses reversible terminators?

Which of the following statements about plasmids is true?

What is the purpose of CsCl density gradient centrifugation in isolating plasmids?

What is the purpose of phenol-chloroform extraction in DNA or RNA purification?

What is the purpose of minipreparation of plasmid DNA?

Which detection method in real-time PCR involves the use of a dye that intercalates with double stranded DNA and causes fluorescence?

Which detection method in real-time PCR involves the use of a probe that is cleaved by the polymerase enzyme, resulting in increased fluorescence?

Which detection method in real-time PCR involves the use of two sequence-specific oligonucleotide probes labeled with different dyes?

What is the purpose of melting curve analysis in real-time PCR?

Which of the following is a key factor that limits the number of DNA molecules that can be achieved in a PCR reaction?

What is the formula used to calculate the theoretically predicted number of DNA molecules, Nm, at the end of a PCR reaction?

What is the purpose of real-time PCR?

What are the two major detection schemes commonly used to track the progress of a PCR reaction?