p.22
DNA/RNA Extraction Process Steps
What is the expected location of DNA if the binding buffer did not contain chaotropic salts?
In the flow-through after the wash step.
p.10
RNA Extraction Process Steps
What is the best practice for isolating RNA from a sample?
Proceed to the lysis step immediately upon disruption of the cells.
p.21
DNA/RNA Extraction Process Steps
Where is DNA found immediately after the wash step?
Still bound to the column.
p.22
DNA/RNA Extraction Process Steps
What is the consequence of preparing wash buffer with water instead of ethanol?
DNA may not be effectively washed away and could remain on the column.
p.4
Chemical Differences Between DNA and RNA
Why are nucleic acids soluble in water?
Because they are polar molecules and hydrophilic.
p.11
Chemical Differences Between DNA and RNA
Why is it more difficult to chemically separate DNA from RNA?
Because they share similar chemical properties and structures.
p.12
Chemical Differences Between DNA and RNA
What is the charge and polarity of lipids?
Uncharged or weakly charged and Non-polar.
p.18
Chemical Lysis and Purification Methods
How is the wash buffer applied to the silica column?
By centrifugation or vacuum.
p.23
DNA/RNA Extraction Process Steps
What is the consequence of preparing wash buffer with water instead of ethanol?
DNA would be found in the flow-through after the wash step.
p.10
RNA Extraction Process Steps
What is an example of a chemical method to stabilize RNA?
Using RNA stabilizing agents such as RNA later (Invitrogen).
p.17
Chemical Differences Between DNA and RNA
What charge does a silica membrane have in water?
Somewhat negative charge.
p.27
Integrity and Purity Analysis of DNA/RNA
How do we assess the purity and integrity of DNA/RNA samples?
Through methods such as spectrophotometry, gel electrophoresis, and assessing the A260/A280 ratio.
p.4
Chemical Differences Between DNA and RNA
What gives nucleic acids their net negative charge?
The phosphate groups (PO₄²⁻) on the sugar-phosphate backbone.
p.22
DNA/RNA Extraction Process Steps
Where would you expect to find your DNA after completing the isolation protocol if errors were made?
In the flow-through after the wash step.
p.26
Integrity and Purity Analysis of DNA/RNA
How can you determine if your sample did not degrade?
By using agarose gel electrophoresis.
p.10
RNA Extraction Process Steps
What should be done if immediate lysis is not possible?
Preserve RNA within the sample.
p.5
Nucleotide Structure and Composition
What bases are used in DNA?
Adenine, Thymine, Cytosine, Guanine.
p.18
DNA/RNA Extraction Process Steps
What is the wash step in DNA/RNA purification?
The process of applying wash buffer to remove contaminants while maintaining binding conditions.
p.13
DNA/RNA Extraction Process Steps
What is the purpose of treating RNA samples with DNase?
To obtain the highest purity by degrading DNA.
p.17
Chemical Lysis and Purification Methods
How do chaotropic salts affect water molecules during the binding step?
They displace water molecules.
p.3
Nucleotide Structure and Composition
What type of sugar is found in nucleotides?
Pentose (5-carbon) sugar.
p.19
DNA/RNA Extraction Process Steps
What is applied to the column during the elution step?
An aqueous solution (water or buffer).
p.7
Chemical Differences Between DNA and RNA
Is RNA single-stranded or double-stranded?
Single-stranded (but frequently has secondary structure with regions of double-strandedness).
p.18
DNA/RNA Extraction Process Steps
What is the purpose of washing DNA/RNA during purification?
To remove salts and impurities.
p.12
Challenges in RNA Extraction
Why is it more difficult to separate DNA from RNA compared to lipids or proteins?
DNA and RNA are chemically more similar to each other than to other macromolecules.
p.13
Challenges in RNA Extraction
Why is it more difficult to chemically separate DNA from RNA compared to lipids or proteins?
The text does not specify, but it implies that the chemical properties of DNA and RNA make their separation challenging.
p.10
RNA Extraction Process Steps
What is one physical method to preserve RNA?
Flash freeze in liquid nitrogen.
p.5
Nucleotide Structure and Composition
What bases are used in RNA?
Adenine, Uracil, Cytosine, Guanine.
p.1
Integrity and Purity Analysis of DNA/RNA
What is the purpose of analyzing the integrity and purity of DNA/RNA extractions?
To ensure the quality of the samples for further experiments.
p.16
Integrity and Purity Analysis of DNA/RNA
What does the pellet contain after centrifugation in DNA/RNA purification?
Lipids, proteins, and other insoluble cell debris.
p.8
DNA/RNA Extraction Process Steps
What is the purpose of washing the DNA during extraction?
To remove salts and impurities.
p.24
Integrity and Purity Analysis of DNA/RNA
What is a significant limitation of using UV absorbance at 260 nm?
It cannot distinguish between DNA and RNA.
p.19
DNA/RNA Extraction Process Steps
How is the aqueous solution forced through the column?
By centrifugation or vacuum.
p.2
Major Biomolecules and Their Subunits
What are the subunits of Carbohydrates?
Sugars (monosaccharides).
p.18
Chemical Lysis and Purification Methods
What type of buffer is applied during the wash step?
A wash buffer, often ethanol-based.
p.16
DNA/RNA Extraction Process Steps
What is the purpose of clearing the lysate in DNA/RNA purification?
To remove cell debris such as proteins, lipids, and saccharides.
p.23
DNA/RNA Extraction Process Steps
Why would DNA be in the flow-through after the wash step if wash buffer was prepared with water?
Binding conditions (dehydration) were not maintained during the wash step.
p.27
Separation Techniques for DNA and RNA
How do the chemical properties of DNA and RNA aid in their separation from other macromolecules?
Their charge and polarity allow for selective binding and elution during purification processes.
p.3
Nucleotide Structure and Composition
Where is the phosphate group attached in a nucleotide?
To the 5′ carbon of the sugar.
p.15
Chemical Lysis and Purification Methods
What is a common detergent used in chemical lysis buffers?
Sodium dodecyl sulfate (SDS).
p.24
Integrity and Purity Analysis of DNA/RNA
What is the advantage of using fluorescent dyes for quantifying DNA/RNA?
They are more specific and provide more accurate results.
p.4
Chemical Differences Between DNA and RNA
What is the polarity of nucleic acids?
Nucleic acids are polar molecules.
p.13
DNA/RNA Extraction Process Steps
What enzyme is used to treat DNA samples for purification?
RNase (RNA-degrading enzymes).
p.1
Chemical Differences Between DNA and RNA
What are the chemical differences between DNA and RNA molecules?
DNA contains deoxyribose sugar and thymine, while RNA contains ribose sugar and uracil.
p.13
DNA/RNA Extraction Process Steps
What is the purpose of treating DNA samples with RNase?
To obtain the highest purity by degrading RNA.
p.17
Chemical Lysis and Purification Methods
What role do chaotropic salts play in DNA/RNA purification?
They are essential for allowing the DNA/RNA to bind.
p.5
Nucleotide Structure and Composition
Is RNA self-replicating?
No, it is synthesized from DNA.
p.8
DNA/RNA Extraction Process Steps
What must be done after lysing the cells in DNA extraction?
Clear the lysate of cell debris (proteins, lipids, saccharides).
p.8
DNA/RNA Extraction Process Steps
What is a common characteristic of all DNA extraction techniques?
They follow the same basic workflow.
p.7
Chemical Differences Between DNA and RNA
Which bases are used in RNA?
Adenine (A), Cytosine (C), Guanine (G), Uracil (U).
p.19
DNA/RNA Extraction Process Steps
What is the first step in collecting DNA/RNA during purification?
Transferring the column to a clean microcentrifuge tube.
p.25
Integrity and Purity Analysis of DNA/RNA
What are some limitations of using the 260/280 ratio?
Factors like pH of the sample buffer can alter readings; it cannot distinguish DNA from RNA.
p.22
DNA/RNA Extraction Process Steps
What happens if wash buffer is used instead of elution buffer to elute DNA?
DNA would likely remain bound to the column.
p.26
Integrity and Purity Analysis of DNA/RNA
What does genomic DNA look like on an agarose gel?
It appears as a single, large band at the top of the gel.
p.16
DNA/RNA Extraction Process Steps
What physical method is commonly used in kits for DNA/RNA purification?
High-speed centrifugation or filtering to separate cell debris.
p.1
Integrity and Purity Analysis of DNA/RNA
What is the significance of the UV spectroscopy 260/280 ratio?
It helps assess the purity of DNA/RNA by indicating protein contamination.
p.8
DNA/RNA Extraction Process Steps
What is one application of isolated DNA?
Polymerase chain reaction (PCR).
p.8
DNA/RNA Extraction Process Steps
What is the final step in the DNA extraction process?
Rehydrate and concentrate the DNA.
p.8
DNA/RNA Extraction Process Steps
What is one use of isolated DNA in research?
Analyzing protein-DNA interactions.
p.9
RNA Extraction Process Steps
What is the purpose of washing the RNA during extraction?
To remove salts and impurities.
p.24
Integrity and Purity Analysis of DNA/RNA
What is an example of a UV spectrophotometer used for nucleic acid quantification?
Nanodrop (Thermo Scientific).
p.23
DNA/RNA Extraction Process Steps
What happens if the binding buffer does not contain chaotropic salts?
DNA would be found in the flow-through after the binding step.
p.10
RNA Extraction Process Steps
Why is it essential to proceed quickly to the lysis step?
To prevent RNA degradation and alteration of specific RNA levels.
p.23
DNA/RNA Extraction Process Steps
What occurs if DNA is not rehydrated and binding conditions are not reversed?
DNA would remain bound to the column.
p.26
Integrity and Purity Analysis of DNA/RNA
How many distinct bands should you see for rRNA on an agarose gel?
2-3 distinct bands representing the small and large subunits of the ribosome.
p.1
Integrity and Purity Analysis of DNA/RNA
What method can be used to analyze DNA/RNA integrity?
Agarose gel electrophoresis.
p.17
Chemical Lysis and Purification Methods
What does the 'cation bridge' do in the binding process?
It allows negatively charged nucleic acids to bind to the silica membrane.
p.3
Major Biomolecules and Their Subunits
What are nucleic acids made of?
Polymers made of repeating nucleotide monomers.
p.9
Challenges in RNA Extraction
How do environmental conditions affect RNA extraction?
Cellular gene expression profiles are rapidly altered in response to changing environmental conditions.
p.9
RNA Extraction Process Steps
What is the first step in the RNA extraction process?
Lyse (break open) the cells.
p.9
RNA Extraction Process Steps
What is the final step in the RNA extraction process?
Rehydrate and concentrate the RNA.
p.25
Integrity and Purity Analysis of DNA/RNA
What is the formula to determine the 260/280 ratio?
Divide the absorbance of the sample at 260 nm by the absorbance at 280 nm.
p.25
Integrity and Purity Analysis of DNA/RNA
What method can be used to check for DNA contamination in RNA samples?
PCR (Polymerase Chain Reaction).
p.11
DNA/RNA Extraction Process Steps
How does the difficulty of separating DNA and RNA compare to other cellular components?
It is more difficult than separating lipids or proteins.
p.13
DNA/RNA Extraction Process Steps
What enzyme is used to treat RNA samples for purification?
DNase (DNA-degrading enzymes).
p.1
Downstream Techniques for Purified DNA/RNA
What is a downstream technique that requires purified DNA or RNA?
Polymerase Chain Reaction (PCR).
p.8
DNA/RNA Extraction Process Steps
What is the first step in the DNA extraction workflow?
Lyse (“break open”) the cells.
p.24
Integrity and Purity Analysis of DNA/RNA
What is a major advantage of using UV absorbance for quantifying DNA/RNA?
It is simple and requires minimal sample preparation.
p.15
Chemical Lysis and Purification Methods
Name two examples of chaotropic salts used in lysis buffers.
Guanidium thiocyanate and guanidium HCl.
p.7
Chemical Differences Between DNA and RNA
Which bases are used in DNA?
Adenine (A), Cytosine (C), Guanine (G), Thymine (T).
p.25
Integrity and Purity Analysis of DNA/RNA
What does it indicate if amplification is observed during PCR of an RNA sample?
It indicates the presence of DNA contamination in the sample.
p.23
DNA/RNA Extraction Process Steps
What is the result of using wash buffer instead of elution buffer to elute DNA?
DNA would remain bound to the column.
p.26
Integrity and Purity Analysis of DNA/RNA
What does a smear on an agarose gel indicate?
That the sample has degraded.
p.27
Chemical Differences Between DNA and RNA
What are the chemical properties of DNA and RNA?
They differ in charge, polarity, and structure, with DNA being double-stranded and RNA being single-stranded.
p.1
DNA/RNA Extraction Process Steps
What is the chemical rationale behind the DNA/RNA extraction process?
It involves breaking down cell membranes and separating nucleic acids from proteins and other cellular components.
p.3
Nucleotide Structure and Composition
What are the three parts of a nucleotide?
Pentose sugar, phosphate group, and nitrogenous base.
p.3
Nucleotide Structure and Composition
Where is the nitrogenous base attached in a nucleotide?
To the 1′ carbon of the sugar.
p.15
Chemical Lysis and Purification Methods
What must be done to alkaline solutions after lysis?
They must be neutralized.
p.24
Integrity and Purity Analysis of DNA/RNA
What is a disadvantage of using fluorescent dyes for quantification?
Some sample preparation is required and a standard curve must be generated.
p.2
Major Biomolecules and Their Subunits
Where are Carbohydrates primarily found?
Cytoplasm, modified proteins, cell surface.
p.27
Chemical Lysis and Purification Methods
What are the basic chemistry principles underlying column-based purification?
The principles include binding of nucleic acids to the column, washing to remove impurities, and elution to recover purified DNA/RNA.
p.9
RNA Extraction Process Steps
What are some uses of isolated RNA?
Gene expression analysis, understanding RNA processing, transcriptomics, and RNA structural analysis.
p.19
DNA/RNA Extraction Process Steps
What is the purpose of the elution step in DNA/RNA purification?
To release hydrophilic DNA/RNA molecules from the silica membrane.
p.9
RNA Extraction Process Steps
What is done after lysing the cells in RNA extraction?
Clear the lysate of cell debris (proteins, lipids, saccharides).
p.15
Chemical Lysis and Purification Methods
Why are chaotropic salts considered essential in kit chemistry?
They play a crucial role in disrupting molecular interactions.
p.25
Integrity and Purity Analysis of DNA/RNA
How can you determine if your DNA/RNA is contaminated with other substances?
By checking the purity using UV absorbance.
p.9
Challenges in RNA Extraction
What is a challenge of RNA extraction related to its nature?
RNA is a transient molecule that is rapidly synthesized and degraded by cells.
p.9
Challenges in RNA Extraction
What are RNases and why are they a challenge in RNA extraction?
RNases are abundant in cells and the environment, making it easy to contaminate and degrade RNA samples.
p.15
Chemical Lysis and Purification Methods
What role do chaotropic salts play in DNA/RNA purification?
They disrupt hydrogen bonding of water molecules.
p.9
RNA Extraction Process Steps
What is important for RNA sample collection and storage?
Proper techniques to prevent degradation and contamination.
