p.7
Beer’s Law and DNA Concentration Calculation
How do you calculate DNA concentration from A260 value?
Multiply the A260 value by 50 μg/mL.
p.7
Absorbance Measurements at 260 nm
What does an A260 value of 1.0 indicate for a DNA solution?
It corresponds to a DNA concentration of 50 μg DNA/mL.
p.17
Ligation Reactions in Molecular Biology
What is generated as a result of ligation?
An intact sugar-PO4 backbone.
p.13
Beer’s Law and DNA Concentration Calculation
How do you calculate the concentration of DNA in μg/mL from the A260 value?
Concentration (μg/mL) = A260 value × dilution factor × 50.
p.10
Beer’s Law and DNA Concentration Calculation
What is the typical extinction coefficient for DNA at 260 nm?
Approximately 50 µg/mL per absorbance unit.
p.18
Ligation Reactions in Molecular Biology
What happens to the adenyl group in the second step of the ligation reaction?
The adenyl group is transferred from the enzyme to the 5’ - PO4 group of the DNA, thereby activating it.
p.18
Ligation Reactions in Molecular Biology
What is formed during the nucleophilic attack in the third step of the ligation reaction?
A phosphodiester bond is formed with simultaneous release of AMP.
p.18
Ligation Reactions in Molecular Biology
What is released during the formation of the phosphodiester bond?
AMP and PO4 are released.
p.2
CTAB Method for DNA Extraction
What types of ethanol concentrations are used in the CTAB method?
70%, 80%, and 100% ethanol.
p.4
Spectrophotometric Analysis of DNA
What does a drop in absorbance at 230 nm indicate in a dsDNA scan?
The presence of contaminants such as phenol or other organic compounds.
p.19
Ligation Reactions in Molecular Biology
Why is the ligase reaction considered inefficient?
Because the DNA ends must remain together for a sufficient time, which can be challenging.
p.1
Genomic DNA Isolation Techniques
What methods can be used for DNA purification?
Phenol-chloroform extraction, ethanol precipitation, and column-based methods.
p.2
CTAB Method for DNA Extraction
What is the purpose of NaCl in the CTAB method?
To help stabilize the DNA during extraction.
p.5
Beer’s Law and DNA Concentration Calculation
What does 'A' represent in the equation I₀ A = εcl?
'A' represents the absorbance of the sample.
p.14
Ligation Reactions in Molecular Biology
What is produced as a result of the ligase-catalyzed reaction?
An intact sugar-phosphate backbone.
p.20
Molar Ratios in DNA Ligation
What does 'Kb' stand for in the context of DNA?
Kilobase, a unit of measurement for DNA length.
p.13
Absorbance Measurements at 260 nm
What is the dilution factor for the single-stranded DNA sample?
1000 μL total volume with 10 μL of DNA.
p.4
Spectrophotometric Analysis of DNA
What is the significance of the peak absorbance at 260 nm in dsDNA?
It indicates the concentration of DNA in the sample.
p.1
Genomic DNA Isolation Techniques
What role do enzymes play in genomic DNA isolation?
They help break down proteins and other cellular components.
p.5
Beer’s Law and DNA Concentration Calculation
What does 'l' denote in the equation I₀ A = εcl?
'l' denotes the path length of the cuvette.
p.17
Role of Ligase Enzymes
What type of DNA damage can ligase activity repair in vivo?
Single stranded nicks in duplex DNA.
p.1
Genomic DNA Isolation Techniques
Why is purification important after DNA isolation?
To remove contaminants that may inhibit downstream applications.
p.19
Optimal Conditions for Ligase Activity
How does temperature affect the movement of particles in a ligase reaction?
At lower temperatures, particle movement is slower, allowing DNA ends to collide and stay together longer.
p.16
Ligation Reactions in Molecular Biology
What is the significance of the enzyme-AMP complex in ligation?
It is involved in the ligation process.
p.16
Role of Ligase Enzymes
What is the difference between E. coli ligase and T4 ligase regarding blunt ends?
E. coli ligase does not ligate blunt ends, while T4 ligase does.
p.5
Beer’s Law and DNA Concentration Calculation
What does 'ε' signify in the equation I₀ A = εcl?
'ε' signifies the molar absorptivity coefficient.
p.8
Absorbance Measurements at 260 nm
What is the relationship between DNA concentration and absorbance at 260 nm?
It is a linear relationship up to an A260 value of approximately 2.00.
p.11
Beer’s Law and DNA Concentration Calculation
What is the relationship between absorbance and concentration in DNA solutions?
Absorbance is directly proportional to concentration according to Beer’s Law.
p.18
Ligation Reactions in Molecular Biology
What is the first step in the ligation reaction?
Formation of enzyme + nucleotide intermediate through the transfer of an AMP from ATP/NAD to the epsilon-amino group of a lysine residue in the ligase enzyme.
p.9
Beer’s Law and DNA Concentration Calculation
How do you calculate the DNA concentration from the absorbance at 260 nm?
Using the formula: DNA concentration (µg/mL) = A260 × dilution factor × 50.
p.18
Ligation Reactions in Molecular Biology
What is the role of the 3’OH group in the ligation reaction?
It performs a nucleophilic attack on the activated PO4 group of the DNA.
p.5
Beer’s Law and DNA Concentration Calculation
In the equation I₀ A = εcl, what does 'I₀' stand for?
'I₀' represents the intensity of incident light.
p.12
DNA Concentration Calculation
How do you convert mM to pmol/μL?
Multiply the concentration in mM by 1,000.
p.1
Genomic DNA Isolation Techniques
What is the primary purpose of genomic DNA isolation?
To extract and purify DNA from cells for analysis.
p.1
Genomic DNA Isolation Techniques
What is a key step in the genomic DNA isolation process?
Cell lysis to release DNA.
p.9
Spectrophotometric Analysis of DNA
What is the A260/A280 ratio of the purified DNA?
1.70 (calculated as 0.550 / 0.324).
p.2
CTAB Method for DNA Extraction
What is the role of high-salt TE buffer in the CTAB method?
To maintain the integrity of the DNA during extraction.
p.14
Ligation Reactions in Molecular Biology
What reaction does ligase catalyze in DNA?
It links the phosphate group from the 5' end of one DNA strand to the hydroxyl group from the 3' end of another.
p.20
Molar Ratios in DNA Ligation
How is the molar mass of insert DNA calculated?
Molar mass of insert DNA required = Kb of insert DNA x ng of vector DNA / Kb of vector DNA.
p.1
Genomic DNA Isolation Techniques
What are common sources for genomic DNA isolation?
Blood, tissues, plants, and bacteria.
p.17
Applications of Single-Stranded DNA
How is ligase activity used in vitro?
To join restriction fragments with compatible vectors having either blunt ends or homologous cohesive ends.
p.10
Beer’s Law and DNA Concentration Calculation
How do you calculate the concentration of a DNA solution from absorbance?
Using the formula: Concentration (mM) = Absorbance / Extinction Coefficient.
p.2
CTAB Method for DNA Extraction
What is the primary component of the CTAB extraction solution?
CTAB (Cetyltrimethylammonium bromide).
p.6
Spectrophotometric Analysis of DNA
What is the significance of a G+C DNA content of 50% in this context?
It is an assumption for calculating the absorption value.
p.9
Beer’s Law and DNA Concentration Calculation
What is the dilution factor for the DNA sample?
50 (since 20μL was diluted to 1000μL).
p.5
Beer’s Law and DNA Concentration Calculation
What does the equation I₀ A = εcl represent?
It represents the relationship between light absorbed by a sample in a cuvette.
p.9
Applications of Single-Stranded DNA
How much plasmid DNA is available for downstream processing after dilution?
Calculate using the concentration obtained from the 50μL plasmid prep.
p.14
Ligation Reactions in Molecular Biology
Which ends of the DNA strands are involved in the ligase reaction?
The 5' end of one strand and the 3' end of the other strand.
p.17
Ligation Reactions in Molecular Biology
What reaction does ligation catalyze?
The linking of the phosphate group from the 5' end of one DNA strand to the OH group of another strand.
p.4
Spectrophotometric Analysis of DNA
What does a typical spectrophotometric scan of dsDNA measure?
The absorbance of double-stranded DNA at various wavelengths.
p.6
Spectrophotometric Analysis of DNA
What does the absorption value of 20 for a 1 mg DNA/mL solution represent?
DNA's extinction coefficient.
p.19
Ligation Reactions in Molecular Biology
What is necessary for a successful ligase reaction?
DNA ends must collide and stay together long enough for ligase to join them.
p.19
Optimal Conditions for Ligase Activity
At what temperature is the conventional ligase reaction conducted?
At a low temperature of 4 °C.
p.10
Beer’s Law and DNA Concentration Calculation
How do you convert µg/mL to millimolarity (mM) for DNA?
Use the formula: Concentration (mM) = (Concentration in µg/mL) / (DNA molecular weight in g/mol).
p.21
Molar Ratios in DNA Ligation
How do you calculate the amount of PCR product needed for a 3:1 molar ratio?
Calculate the molar concentration of the vector and then determine the corresponding amount of PCR product to achieve the desired ratio.
p.9
Spectrophotometric Analysis of DNA
What does an A260/A280 ratio of around 1.8 indicate?
It indicates high purity of DNA.
p.5
Beer’s Law and DNA Concentration Calculation
In the equation I₀ A = εcl, what does 'c' represent?
'c' represents the concentration of the sample.
