How do you calculate DNA concentration from A260 value?
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Multiply the A260 value by 50 μg/mL.
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How do you calculate DNA concentration from A260 value?
Multiply the A260 value by 50 μg/mL.
What does an A260 value of 1.0 indicate for a DNA solution?
It corresponds to a DNA concentration of 50 μg DNA/mL.
What is the maximum A260 value for the linear relationship of DNA concentration and absorbance?
Approximately 2.00.
What is generated as a result of ligation?
An intact sugar-PO4 backbone.
How do you calculate the concentration of DNA in μg/mL from the A260 value?
Concentration (μg/mL) = A260 value × dilution factor × 50.
What is the symbol used to represent DNA's extinction coefficient?
E, e, or ε.
What is the typical ratio of absorbance at 260 nm to 280 nm for pure dsDNA?
Approximately 1.8.
What is the typical extinction coefficient for DNA at 260 nm?
Approximately 50 µg/mL per absorbance unit.
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.
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.
What is released during the formation of the phosphodiester bond?
AMP and PO4 are released.
What types of ethanol concentrations are used in the CTAB method?
70%, 80%, and 100% ethanol.
What is the optimum molar ratio of insert to vector for an efficient ligase reaction?
3:1.
What is the absorption value (A260) of a 1 mg/mL DNA solution at neutral pH?
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.
Why is the ligase reaction considered inefficient?
Because the DNA ends must remain together for a sufficient time, which can be challenging.
What methods can be used for DNA purification?
Phenol-chloroform extraction, ethanol precipitation, and column-based methods.
What is the purpose of NaCl in the CTAB method?
To help stabilize the DNA during extraction.
What is the ratio of chloroform to octanol or isoamyl alcohol used in the CTAB method?
24:1 (v/v).
What does 'A' represent in the equation I₀ A = εcl?
'A' represents the absorbance of the sample.
What is produced as a result of the ligase-catalyzed reaction?
An intact sugar-phosphate backbone.
What does 'Kb' stand for in the context of DNA?
Kilobase, a unit of measurement for DNA length.
What is the dilution factor for the single-stranded DNA sample?
1000 μL total volume with 10 μL of DNA.
What is the significance of the peak absorbance at 260 nm in dsDNA?
It indicates the concentration of DNA in the sample.
What role do enzymes play in genomic DNA isolation?
They help break down proteins and other cellular components.
What is the absorbance reading at 260 nm for the diluted DNA sample?
0.550
What is the desired insert to vector molar ratio for the ligation?
3:1.
How much vector is used in the ligation?
50 ng of 3.0 kb vector.
What is the optimal activity temperature for ligase enzymes?
25 °C.
What does 'l' denote in the equation I₀ A = εcl?
'l' denotes the path length of the cuvette.
What is the absorbance of the DNA solution at 260 nm?
1.00.
What is the A260 value obtained for the diluted single-stranded DNA sample?
0.125.
What type of DNA damage can ligase activity repair in vivo?
Single stranded nicks in duplex DNA.
What is the absorbance of the DNA solution at 260 nm?
0.212.
Why is purification important after DNA isolation?
To remove contaminants that may inhibit downstream applications.
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.
If the absorbance is 0.212, what is the concentration of the DNA solution in µg/mL?
10.6 µg/mL (0.212 x 50).
What is the concentration of 2-Mercaptoethanol used in the CTAB precipitation solution?
2% (v/v).
What is the significance of the enzyme-AMP complex in ligation?
It is involved in the ligation process.
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.
What does 'ε' signify in the equation I₀ A = εcl?
'ε' signifies the molar absorptivity coefficient.
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.
What is the relationship between absorbance and concentration in DNA solutions?
Absorbance is directly proportional to concentration according to Beer’s Law.
What is the concentration of the single-stranded DNA sample in μg/mL?
6.25 μg/mL.
What is the formula that describes the relationship between absorbance, concentration, light path length, and extinction coefficient?
A260 = E260 * l * C.
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.
What is the absorbance reading at 280 nm for the diluted DNA sample?
0.324
How do you calculate the DNA concentration from the absorbance at 260 nm?
Using the formula: DNA concentration (µg/mL) = A260 × dilution factor × 50.
What is the role of NAD in E. coli ligase activity?
It acts as a cofactor.
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.
In the equation I₀ A = εcl, what does 'I₀' stand for?
'I₀' represents the intensity of incident light.
How do you express the concentration of DNA in this problem?
In millimolarity.
What is the concentration of the DNA solution in mM?
0.03 mM.
How do you convert mM to pmol/μL?
Multiply the concentration in mM by 1,000.
What is the primary purpose of genomic DNA isolation?
To extract and purify DNA from cells for analysis.
At which wavelength is dsDNA typically measured?
260 nm.
What is a key step in the genomic DNA isolation process?
Cell lysis to release DNA.
What organism is associated with the ligase that requires NAD as a cofactor?
E. coli ligase.
How can concentration (C) be calculated using absorbance (A260) and extinction coefficient (E260)?
C = A260 / E260.
Which ligase can ligate blunt ends?
T4 ligase.
What type of bond do ligases form?
Phosphodiester bond.
What is the size of the PCR product to be added?
0.5 kb.
What is the A260/A280 ratio of the purified DNA?
1.70 (calculated as 0.550 / 0.324).
What is the role of high-salt TE buffer in the CTAB method?
To maintain the integrity of the DNA during extraction.
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.
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.
What is the concentration of the DNA solution expressed as pmol/μL?
30 pmol/μL.
What are common sources for genomic DNA isolation?
Blood, tissues, plants, and bacteria.
How is ligase activity used in vitro?
To join restriction fragments with compatible vectors having either blunt ends or homologous cohesive ends.
How do you calculate the concentration of a DNA solution from absorbance?
Using the formula: Concentration (mM) = Absorbance / Extinction Coefficient.
What is the primary component of the CTAB extraction solution?
CTAB (Cetyltrimethylammonium bromide).
What is the significance of a G+C DNA content of 50% in this context?
It is an assumption for calculating the absorption value.
What is the dilution factor for the DNA sample?
50 (since 20μL was diluted to 1000μL).
What does the equation I₀ A = εcl represent?
It represents the relationship between light absorbed by a sample in a cuvette.
How much plasmid DNA is available for downstream processing after dilution?
Calculate using the concentration obtained from the 50μL plasmid prep.
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.
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.
What does a typical spectrophotometric scan of dsDNA measure?
The absorbance of double-stranded DNA at various wavelengths.
What does the absorption value of 20 for a 1 mg DNA/mL solution represent?
DNA's extinction coefficient.
What is necessary for a successful ligase reaction?
DNA ends must collide and stay together long enough for ligase to join them.
What is the cofactor required by T4 ligase?
ATP.
At what temperature is the conventional ligase reaction conducted?
At a low temperature of 4 °C.
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).
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.
What does an A260/A280 ratio of around 1.8 indicate?
It indicates high purity of DNA.
In the equation I₀ A = εcl, what does 'c' represent?
'c' represents the concentration of the sample.
