Randy Masco

BIOL 3612 – Lab for Biochemistry

TA Oljora Rezhdo
5 October 2017

Q1) My group isolated strawberry DNA utilizing the precipitation method. We were able to use
the Nanodrop to measure its concentration three times, which were calculated as 3.8 ng/ul, 3.5
ng/uL and 3.6 ng/uL. These readings were all produced by the same Nanodrop with the same
person administering the droplets. The reproducibility, defined as consistency of measurements
over multiple instrumental readings, was generally acceptable, with no deviations greater than
0.3 ng/uL, and therefore the average would be a viable method for calculating the yield of our
DNA preparation. This is usable because the DNA concentrations all lie in a range of 0.3 ng/uL
and have no major discrepancies.

Q2) Utilizing the average DNA concentration from the three measurements above, calculated as
3.63 ng/uL by summing them together and then dividing by the number of values (3), the total
yield for the precipitation method is 1800 ng. This was deduced by multiplying the isolated DNA
concentration (3.63 ng/uL) by the final suspension volume of the DNA (500 uL). Expressed as a
ratio of the original starting strawberry material, measured as 54.5 grams, the total yield is 33
ng/g of solid strawberry mass. Using the same method of yield calculation, the DNA isolated
using the column method produced a yield of 978 ng/g of starting material. This was similarly
calculated by multiplying their DNA concentration (4.5 ng/uL) by their volume (100 uL) and
then dividing that total (450 ng) by the amount of strawberry material they started with,
described as 0.46 g.
Method of Isolation Mass of DNA (ng) Total Yield (ng/g)
Isolation by Precipitation 1800 ng 33 ng/g
Isolation by Column 450 ng 978 ng/g

The method that yielded a higher total mass of DNA was the precipitation method. This can be
explained by referring back to the amount of starting DNA material, measured as 54.52 grams, a
mass more than 100 times the starting material of the column method, measured as 0.46 grams.
Because there was so much more to work with, the likelihood of more material being retained
throughout the method is higher. This can also be related back to the fact that the precipitation
method was much coarser, requiring little purification steps, and thus there was less opportunity
for potential product to be removed. There is also a possibility that other biomolecules such as
proteins and RNA were not fully removed from the solution measured by Nanodrop, due to the
aforementioned lack of rigorous purification, and therefore contribute to this larger mass.
The column purification method yielded the larger ratio of isolated DNA to starting
material, greater than that of the precipitation method by a factor of almost 30. This is due to the
more intense and precise methods of purification used to isolate the DNA, specifically the
addition of multiple lysis and wash buffers which are engineered to separate DNA from other
biomolecules as well as the use of Qiagen spin column which, in combination with the RNAse,
lysis and wash buffers, has a high affinity for DNA and can more readily eliminate unwanted
biomolecules and reagents through multiple rounds of centrifugation.
Taking into account the methodology of the precipitation procedure I performed, some
steps in repeat experimentation to improve my yield would be to more rigorously mash the
strawberries to obtain a solution that can more easily pass through the cheesecloth and use an
electronic micropipette to more slowly and consistently administer the ethanol and therefore
generate a more palpable DNA aggregate.

Q3) As compared to the “ideal” Nucleic Acid spectrum displayed in the NanoDrop Nucleic Acid
Handbook, our measured precipitation spectrum is radically different. The ideal spectrum
displays a dip of ~6 absorbance units from 220 nm to 230 nm, followed by a rise of ~20
absorbance units to a peak at 260 nm. The spectrum for our isolated DNA gradually dropped
from a ~.2 absorbance to a ~0.05 absorbance between 220 nm and 245 nm, and then never rose
to a peak, instead gradually tapering off to a ~.02 absorbance across the 250 to 320 nm range. It
did not adhere to the expected 280 nm linear stretch exhibited by the ideal spectrum as well. In
terms of the spectrum generated by column purification, the visualization is close to a downslope
hill, similarly starting at 220 nm on a higher 0.3 absorbance and then going gradually straight
down until tapering off at ~270 nm.
As deduced from the troubleshooting section of the handbook, common contaminant
absorbances are usually present between the 240-270 nm wavelength range, such as in phenol
and guanidine isothiocyanate. In the cases of Guanidine HCl and EDTA, they present a
characteristic drop from 220 nm to ~270 nm, granting no absorption ability past these values.
The ability to visualize the entire absorption spectra is useful for this reason, as it allows
researchers to possibly deduce contaminants and thus corrections from their obtained spectra. If a
sample deviates from the expected nucleic acid absorbance pattern, they can analyze the
deviations and the absorbance expectancies of other compounds utilized in the method and make
inferences as to which could possibly be interfering with the desired product.
DNA isolated through the precipitation method has a high likely of being contaminated
with RNA due the lack of RNA-lysis measures utilized through the procedure. The only
measures of separation used were an ethanol treatment which has an affinity for both DNA and
RNA, therefore making it very possible for RNA to contaminate isolated DNA. The column
purification method has a much smaller chance of being contaminated with RNA because of the
RNAse treatment the sample receives early on in the method, an enzyme that specifically lyses
DNA from RNA. It would be difficult to distinguish an RNA absorbance spectrum from a DNA
absorbance spectrum because there are no definitive differences in the peaks they exhibit when
analyzed by the NanoDrop, as both are nucleic acids and behave similarly when subjected to the
light absorption.

Q4)
Method Measurement # A260 (AU) A280 (AU) A260/A280 A260/A230 Conc. (ng/uL)
Precipitation 1 0.08 0.05 1.55 0.46 3.8
2 0.07 0.04 1.82 0.49 3.5
3 0.07 0.05 1.47 0.46 3.6
Column 1 0.10 0.08 1.14 0.42 4.9
2 0.08 0.06 1.34 0.43 4.1

Compared to the ‘accepted’ A260/A280 and A260/A230 ratios in the NanoDrop Nucleic
Acid Handbook, stated as around 1.8 and 1.8-2.2 respectively, the A260/A280 readings fell short
by approximately ~0.3 of the expected ratio for the precipitation method readings – aside from
reading 2, which correctly aligns with 1.8 – and by ~0.8 for the column purification DNA. In
terms of the A260/A230 ratio, all of the measurements collectively fell short by approximately
1.5.
 The concentrations derived from the Nanodrop are calculated using the Beer-Lambert
law as outlined in the handbook. This equation states that the concentration in ng/uL is calculated
by first multiplying the absorbance of the sample (calculated as the absorbance at 260 nm, then
corrected through a baseline analysis of the 340 nm absorbance and a selected analysis constant),
and the extinction coefficient (50 ng-cm/uL for dsDNA) and then dividing the product by the
pathlength of the nanodrop in centimeters. In this equation, the concentration is directly
proportional to the absorbance (with the extinction coefficient acting as the proportionality
constant), while the path length is inversely proportional.
If a pure double-stranded DNA sample and RNA sample had the same A260 reading, the
DNA sample would produce a higher concentration because it’s extinction coefficient is 50 ng-
cm/uL while RNA’s extinction coefficient is 40 ng-cm/uL. Because in this instance the A260
reading is the proportionality constant and the extinction coefficient is directly proportional to
the concentration, the compound with the larger extinction coefficient would produce a larger
concentration. However, if the DNA was single-stranded, RNA would produce a larger
concentration because single stranded DNA has an extinction coefficient of only 33 ng-cm/uL.

Q5) Salt and ethanol both play a role in reducing the polarity of DNA molecules and thus
making them more hydrophobic, allowing them to be more easily extractable from the
strawberry-pulp solution. Positive sodium ions stabilize the negatively-charged phosphate
backbone of DNA that tends to denature in aqueous solutions, allowing it to develop some
insolubility; ethanol provides an easier pathway for these sodium ions to interact with the
backbone. In water, DNA is difficult to stabilize through weak interactions because it has a high
dielectric constant and therefore is difficult for charge to travel through, while ethanol has a
much lower dielectric constant and promotes these interactions. These two compounds thus work
in combination to retain a solution the DNA is able to suspend in without completely denaturing.
(Sources: http://bitesizebio.com/253/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/
; https://sciencing.com/sodium-used-dna-extraction-6504902.html )
The widespread popularity of the DNeasy spin columns among lab groups, despite their
high cost and required use of many reagents, can be accounted for by its effectiveness in
purification and the low amount of starting material needed to complete this purification. As
previously stated, the DNA precipitation method utilized a large amount of starting material
(which is inexpensive in terms of strawberries but may be much more expensive for other
experimental compounds) and was coarse in actually providing purified DNA. The Qiagen spin
columns also provide observable contamination removal, with specific buffers and enzymes
utilized to remove specific unwanted biomolecules present in solution. While the traditional
precipitation method is near impossible to quantify in terms of purified DNA obtained, the
Qiagen spin columns provide a verifiable purity that would be more useful in lab settings, as
pure products are necessary to make deductions from assays.

Q6)

1 2 3 4 5
1

Agarose gel: For reference, well 1 remained empty, well 2 contained the DNA ladder, well 3
contained the precipitation method sample, well 4 contained the column purification
sample, and well 5 contained a positive DNA control.
Based on our gel electrophoresis reading, there is no visualization of purified DNA in
either the column chromatography or the precipitation method, most likely due to the very low
concentration of DNA present in both samples to begin with. To calculate the mass of DNA
added for each sample respectively, a method similar to that used in question 2 was used. The
volume of DNA introduced to each well was respectively 13.5 uL for each; using the known
concentration of the precipitation sample, calculated to be 3.6 ng/uL, the mass of precipitation
sample added is the product of the concentration and the volume, amounting to 48.6 ng. The
mass of column purification sample added, following the same logic, would be the known
concentration of the sample, 4.5 ng/uL, multiplied by the amount added to the gel, 13.5 uL. This
product is equal to 60.75 ng, making the mass of column sample added larger than that of the
precipitation sample.
We included a positive control DNA sample in well 5 of our agarose gel, producing a
band of approximately 5000 bp. To calculate the mass of positive control added to the gel, one
must follow the same logic as the previous paragraph by multiplying the concentration of control
by the volume added. The given concentration of the control, given by the TA, is 0.1 mg/mL, or
100 ng/uL, while the volume of control added to the well was determined to be 4.5 uL. The
product of these two values equals 450 ng, the determined mass of control added to the gel.

Q7) I have several suggestions as to how to improve this lab for future experimentation. The first
of which would be to break open the strawberries using a food processor rather than a mortar and
pestle, an appliance freely available in most Northeastern labs (and dining halls). This would
provide a more even mixture of strawberry pulp that could more easily pass through the
cheesecloth, and would ensure that it is ground down to a good consistency; using a more archaic
tool such as a mortar and pestle (which many newer labs have completely phased out) gives
more room for human error.
Another improvement I would suggest is to expand the column purification method with
more buffers and lysis steps to provide a more pure product. Even though the column
purification was more rigorous and was expected to produce a purer sample, in our case, it still
ended up having a very contaminated and muddy emission spectra, and thus still contained some
unwanted molecules. If the sample was subjected to a multiplicity of these buffer, centrifugation
and extraction steps, the endgame purity would likely be much higher.
A final improvement for suggestion for this lab would be to allow for multiple attempts at
running the gel electrophoresis, possibly across multiple weeks. Although many students were
familiar with the technique and accomplished it well, there were several peers in my lab that had
extreme trouble navigating the buffer/gel line and properly administering the dye/sample
combination. As a result, most of the class ended up with faulty or incomplete gel readings and
were put in a more difficult situation in terms of analyzing the method. If more practice was
given attempting this method and navigating that gel/buffer line I’m sure the finished gel
products would have been easier to elucidate.