Lab Report-3
Course Code: BTE 258
Course Title: Lab 3
Experiment No:3
Estimation of quantity and molecular weight of DNA and
RNA from a sample.
Submitted By,
Zubayer Bin Hossain
ID: 22236092
Section: 3
Submitted To,
Tushar Ahmed Shishir
Lecturer
Department of Mathematics and Natural Science
BRAC University
Objective:
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The aim of this experiment is to determine the quantity, concentration, and purity of the DNA
sample that has been provided, the objective of this experiment is to be fulfilled.
Principle:
The estimation of the molecular weight of DNA and RNA using a Nanodrop relies on its ability
to measure the absorbance of nucleic acids at 260 nm. DNA and RNA absorb ultraviolet light at
this wavelength due to their nucleotide bases. The Nanodrop requires only 1-2 µL of the sample,
which is directly applied to its pedestal. Using the absorbance value at 260 nm, the concentration
of nucleic acids is calculated based on pre-defined conversion factors—50 µg/mL for DNA and
40 µg/mL for RNA when the absorbance is 1.
The purity of the sample is assessed through the A260/A280 and A260/A230 ratio, which
indicates contamination by proteins or other impurities; a ratio of approximately 1.8 signifies
pure DNA, while a ratio of about 2.0 suggests pure RNA. The Nanodrop's integrated software
streamlines these calculations and provides results in nanograms per microliter. To estimate the
molecular weight, the concentration determined by the Nanodrop can be multiplied by the known
sequence length of the nucleic acid. For DNA, the molecular weight is approximately 660
Daltons per base pair, and for RNA, it is about 340 Daltons per nucleotide. This process makes
the Nanodrop a convenient and accurate tool for nucleic acid quantification and molecular
weight estimation. (Garcia-Algeria, [Link]., 2020). Through the ratio of A260/280 we can find out
the concentration of experimental nucleic acid.
Concentration (ng/µL)=A260×conversion factor
Apparatus and Reagents:
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Apparatus:
● UV spectrophotometer
Reagents:
● DNA sample
● TE buffer
Procedure:
Spectrophotometer
1) The DNA sample (20 μl) was taken in the TE buffer.
2) The above sample was diluted by a factor of 100, i.e., 20 μl of the sample was taken in
1980 μl of TE buffer.
3) After this, the optical density values at A260 and A280 were taken, and the amount of
DNA recovered was calculated.
4) The following formula was used to determine the concentration of DNA:
Total DNA (ug) =(A260) (50 ug/ml/A260) (100) (0.02 ml) Where 100 was the dilution
factor, and 0.02 ml was the total volume of the DNA.
5) DNA quality measurement is based on the fact that OD at 260 nm is twice that at 280 nm
if the solution contains pure DNA. If there is a contaminant, there is some additional OD,
which decreases the OD ratio between 260 and 280 nm.
6) Clean DNA has an OD260/OD280 between 1.8 and 2.0.
Observation:
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Fig: Presence of Double Stranded DNA by NanoDrop Machine
The A260/280 shows a ratio of 2.17 and in A260/230 shows 1.43. Again for the second sample the
ratios are 2.1 and 1.22.
Result:
In the first semple A260/280260/280260/280 ratio of 2.17 indicates a sample likely contains
RNA, as pure RNA typically shows a ratio close to 2.0, but the slightly higher value could
suggest contamination with phenol or other reagents. The 260/230260/230260/230 ratio of 1.43,
which is lower than the ideal range (2.0–2.2), suggests the presence of organic compounds, salts,
or other impurities that may interfere with the purity of the sample.
In the next one A 260/280260/280 ratio of 2.1 indicates a predominantly RNA sample, as this is
consistent with the expected range for pure RNA, but it may also reflect slight contamination
from phenol or proteins. The 260/230 ratio of 1.22, however, is significantly lower than the ideal
range, suggesting substantial contamination with organic compounds, salts, or other impurities,
which could affect downstream applications.
Discussion:
The results obtained from the Nanodrop analysis of DNA isolated from blood samples reveal key
insights into the purity and potential contaminants present in the samples. In the first sample, the
260/280260/280 ratio of 2.17 is higher than the typical value of ~1.8 expected for pure DNA,
suggesting that the sample is likely contaminated with RNA, as RNA exhibits a higher
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260/280260/280 ratio near 2.0. Additionally, the 260/230260/230 ratio of 1.43, which falls below
the optimal range of 2.0–2.2, indicates the presence of contaminants such as organic compounds,
phenol, or residual salts from the extraction process. These impurities can interfere with
downstream applications like PCR or sequencing by affecting enzymatic reactions and the
accuracy of quantification.
For the second sample, the 260/280260/280 ratio of 2.1 further supports the presence of RNA
contamination, as it exceeds the expected range for DNA purity. However, the 260/230260/230
ratio of 1.22 suggests a higher degree of contamination with organic solvents or salts than the
first sample. Such impurities are commonly associated with insufficient washing steps during
DNA isolation or the incomplete removal of phenol or ethanol used during the extraction
process. Given that blood was used as the source for DNA isolation, these issues might arise
from the complex matrix of blood, which contains proteins, lipids, and other components that
can co-isolate with nucleic acids if the purification process is not optimized. Addressing these
challenges requires revisiting the extraction protocol, with particular attention to the removal of
RNA (e.g., using RNase treatment) and enhancing the cleanup steps to minimize carryover of
organic and salt contaminants.
Precautions:
● Care should be taken while diluting the DNA sample.
● Thorough washing steps must be performed to eliminate residual salts, phenol, and other
organic solvents that may compromise the quality of the isolated DNA.
● Samples should be handled carefully in a clean and controlled environment to minimize
the risk of environmental contamination.
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Reference:
García-Alegría, A. M., Anduro-Corona, I., Pérez-Martínez, C. J., Corella-Madueño, M. a. G.,
Rascón-Durán, M. L., & Astiazaran-Garcia, H. (2020). Quantification of DNA through
the NanoDrop Spectrophotometer: Methodological Validation Using Standard Reference
Material and Sprague Dawley Rat and Human DNA. International Journal of Analytical
Chemistry, 2020, 1–9. [Link]
