Molecular Biology and Genetics Lab

Spring 2021

ASSIGNMENT 2
EXP 6 & 7

Ela ERCİYES
Student ID: 0076173

MBGE101L (LAB B)
Date of experiment: 12-19.04.2021
Assignment submission date: 27.04.2021
Instructor: Billur Çelebi ERGİN
Q1-

Organic extraction principle: This method is historically the most common method of
extracting genetic material from organisms. It involves homogenization of the sample being
analyzed in a solution containing phenol-usually phenol-chloroform. This phenol-chloroform
mixture is insoluble in water, meaning that centrifugation results in the formation of two distinct
phases.

The dense phase at the bottom of the tube after centrifugation contains denatured proteins, and
the aqueous phase on top contains nucleic acids. The extraction of nucleic acids using this
method is pH dependent, with a pH greater than 7 enabling both DNA and RNA to be dissolved
in the aqueous phase. In contrast, at acidic pH values, DNA is inclined to denature and
precipitate into the dense organic phase.

Once the two phases form, the aqueous phase is carefully pipetted out and the genetic material is
precipitated using alcohol.

Silica column technology: This technique relies on spin columns containing silica in order to
bind nucleic acids in the sample. In order to obtain the nucleic acids, the samples are lysed in
buffered solutions containing RNase inhibitors (since the main goal is extracting RNA). These
lysates are then subjected to centrifugal force and passed through silica membranes at the
appropriate pH so that the RNA can bind to the membrane.

Comparison of these two extraction procedures:

 Organic extraction is applicable to both larger samples like tissues as well as smaller
samples such as cell cultures. In contrast, silica column technology is not able to process
large amounts of sample since the membrane can get clogged.
 Organic extractions can be labor-intensive when performed manually, while silica
column technology makes it simple to do large-scale processing.
 While silica column technologies do not require the usage of harsh chemicals, phenol-
chloroform is used for organic extraction, meaning that great care must be taken in order
to dispose of the hazardous chemical waste appropriately. (1)

Q2- Prokaryotic organisms do not contain any membrane-bound organelles, and thus their
genetic material is loose in the cytoplasm. Additionally, this genetic material is circular and not
packed tightly with histone proteins as opposed to eukaryotic cells. In eukaryotes, DNA is
wrapped in histone proteins and is found within the nucleus. Additionally, eukaryotes also have a
circular DNA founds in their mitochondria and (if present) chloroplasts. This is why, when
extracting DNA from prokaryotic organisms, it is enough to lyse the cell wall and cell membrane
in order to have access to the genetic material. On the other hand, in order to extract DNA from a
eukaryotic organism, one must also lyse the nucleus to be able to access the contents within.

Q3- There are multiple ways to determine DNA yield and purity, two of which are absorbance
and gel electrophoresis. In the absorbance method, the prepared samples are placed into Uv-
transparent cuvettes and then subjected to UV light under a spectrophotometer. Afterwards, the
absorbances at 260nm (where DNA most strongly absorbs light) and 280nm (where aromatic
amino acids are at maximum absorption) are measured, and the following formula is used to
calculate the concentration after the measurements are adjusted for turbidity:

Concentration (µg/ml) = (A260 reading – A320 reading) × dilution factor × 50µg/ml

In order to calculate the total yield:

DNA yield (µg) = DNA concentration × total sample volume (ml)

The yield shows how effective the extraction was, and how well it was able to be purified.

Fluorescence methods ar eyet another popular protocol used when determining DNA yield due to
the wide availability of fluorescent dyes able to bind to DNA. Fluorescence methods are more
sensitive than absorbance methods, especially for samples of lesser concentrations. In this
method, a DNA-binding dye is used and bound to the appropriately prepared DNA samples.
Afterwards, the excitation and emission values for the fluorescent dye chosen is evaluated and a
fluorometer is used to detect the dye. The data obtained from the fluorometer is then compared to
a standard curve generated from DNA samples of known concentrations, and the concentration
as well as yield of the sample is detected.

Q4- In order to estimate the size of the DNA molecules in gel electrophoresis, their migration
speeds (or how far they are able to go in the gel in a given time frame where voltage is applied)
is used. Since agarose gel forms a complex mesh, molecules will have to find their way through
this structure in order to move ahead from the wells. The larger a sample is (large meaning more
base pairs) , the more difficult it will become for it to run on the gel. Therefore, the DNA
samples which move the farthest are classified as the smallest while the ones closest to the
original wells are said to be the largest. A reference can be used to determine how many base
pairs a given sample includes based on its position on the gel. (3)

Q5-

Q6-
Proteinase K is a proteolytic enzyme commonly used in molecular biology laboratories. The "K"
in this enzyme's name refers to the fact that it is able to digest native keratin found in hair.

The usage of Proteinase K in DNA extraction protocols serves to eliminate the contaminating
proteins present within the sample. Some of these proteins are nucleases, which will affect the
experiment negatively by digesting the genetic material wishing to be collected. When
Proteinase K is added to the sample, these nucleases are digested and therefore the genetic
material is protected from degradation. (4)
Q7-

It is important to use centrifugation in DNA extraction protocols as this process ensures thatb we
are able to obtain a clean sample. When compared to other cellular components such as proteins,
DNA has a lower molecular weight, meaning that applying centrifugal force will separate nucleic
acids from unwanted components (the heavier components will form a pellet). (5)

Q8-

Three of the factors which may be manipulated to affect the rate of migration for DNA
molecules in agarose gel electrophoresis:

i. The current and voltage of the run: If the current applied to the system is too high, the
DNA molecules will move faster, the gel will heat up too much and the DNA will smear.
On the other hand, if the current applied is too low, the DNA is unable to run in the gel
and instead diffuses.
ii. Concentration of agarose: As the fragment size of the DNA sample increases, the
concentration of agarose in the gel must be lowered so that the DNA molecules are able
to migrate. If the concentration is kept too high for larger fragments, the genetic material
will not be able to migrate as fast as needed and the results obtained will not be useful.
iii. Temperature of the system: An increase of temperature when performing gel
electrophoresis will increase the strength of the electric field, therefore increasing the
migration speed of DNA. It should be kept in mind that increasing the temperature too
much will cause denaturation, so it is advised to exercise caution when modifying this
factor. (6)

Q9-

SYBR Green:

 Relatively cost-effective
 Simple usage
 Requires usage of green filter
 Non-hazardous, simple disposal
 Non mutagenic when diluted
 Alters electrophoretic mobility (thus affecting DNA size estimations)
 Less photosstable than ethidium bromide

Ethidium Bromide:

 Inexpensive but requires laborious disposal procedures due to its high toxicity.
 More photostable when compared to SYBR green
 Gives more sensitive results than SYBR green
 Mutagen (7)
Q10-

The primary difference between pre-staining and post-staining a gel is the steps of the procedure.

With pre-staining (PS), a miniscule amount of dye is added into the agarose solution before the
gel is poured. Additionally, some dye is added to the running buffer as well. On the other hand,
loading dye is added to the nucleic acid sample before it is pipetted into the agarose gel wells. (8)

Q11- Loading dyes are used in electrophoresis procedures for three main purposes:

i. Since the dye migrates independent of the sample, it is possible to estimate the migration
of proteins and/or DNA and RNA.
ii. The dyes color the samples, which provides visual aid for the process of loading.
iii. Adding a loading dye to the sample causes an increase in density, facilitating even
distribution to the sample wells. (9)

Q12- Since proteins (especially aromatic amino acids) generally absorb light at a wavelength of
280 nm and nucleic acids absorb UV lights at a wavelength of 260 (due to the resonance of
purine and pyrimidine bases), the absorbance values at these two wavelengths provide the
needed insight to determine DNA concentration.

According to the Beer Lambert equation:

A=e*b*c

A = absorbance
e = molar absorptivity coefficient
b = path length in cm
c = molar concentration

Since the path length and coefficient will not change for the data collected at these two
different wavelengths, the ratio between the absorbance values will directly give us the ratio
between the concentration values, meaning that it will be possible to determine the relation
between the protein and nucleic acid concentrations of the sample being assessed.
A1/A2 = (e1 * b1* c1) / (e2 * b2 * c2) --> e and b cancel out

Q13- As a blank reading, it is appropriate to use the buffer present in the samples at the same
composition and pH because this will enable us to "calibrate" our results according to the sample
which does not contain any genetic material. This sample, while miniscule, will still exhibit
absorption so it is necessary to take into account what the absorbance value for the blank sample
is so that accurate estimates can be made for the samples which do contain genetic material.

These two absorbance values can then also be used to calculate the DNA concentration:
Concentration (µg/ml) = (A260 reading – A320 reading) × dilution factor × 50µg/ml
(an A260 of 1.0 at 50µg/ml is accepted as pure.)

(10)

Q14-

In total, the human genome is 3 billion base pairs. Since it is so large (and therefore unable to
migrate quickly through the mesh-like structure of the agarose gel), genomic DNA usually shows
up at the very top of the gel, near the well. For the intact genomic DNA, we would expect to see
a smear on the gel since it contains fragments of different sizes.

The observations made with the gel will also depend on the conformation of the fragments and
their sizes. For example, if the sample were to contain fragments with linear, supercoiled and
nicked; we would expect to see three distinct formations on the gel resulting from the different
speeds at which these fragments move across the gel. However, if all (or a considerable amount)
of the fragments were to be of the same size and same conformation, only one distinct band
formation would be observed.

In order to determine the size of the fragments, size markers are used.

When viewing agarose gel electrophoresis results, it is possible to see one long, continuous
stroke of fluorescence rather than distinct bands. This is called smearing. Smearing can occur as
a result of the following experimental errors:

i. If the gel is prepared improperly, meaning that it is poured unevenly/incorrectly, the

resulting solidification process will not be even and the molecules will smear.
ii. If the wells are overloaded or the sample is not diluted appropriately, the excess amount
can spill out of the well and spread across the gel, once again creating the appearance of a
smudge.
iii. If an incorrect temperature or buffer is chosen for the restriction enzyme present in the
sample, the enzyme may not function as expected and break down the molecule too
much, resulting in a smear.
iv. If the DNA sample being analyzed is contaminated by an unwanted protein, this could
also lead to smearing.

In gel electrophoresis, the amount of bands may exceed the expected amount. This occurs when
the plasmid DNA sample being analyzed is present in multiple different conformations, resulting
in different migration speeds.
These conformations, explained in more detail are:

i. Supercoiled: The circular plasmid is twisted tightly, resulting in a higher density than its
counterparts. This configurational form migrated the fastest and is thus found furthest
from the well.
 ii. Nicked: In the nicked circle configuration, one of the covalent bonds in the plasmids's
sugar-phosphate backbone is broken, rendering the disk "floppy". Plasmid samples in this
configuration move slower than supercoiled samples.
iii. Linearized: Occurs when the plasmid DNA being observed is in a linear state. This
conformation moves at the median speed across the gel. (10)

It is observed that some of the bands are thicker within the DNA ladder well. If a DNA band is
too thick, this might make the end results harder to read; in order to combat this issue the wells
may be loaded less or wider wells may be created. Additionally, when gel electrophoresis is
performed, some of the bands may be observed thicker and more fluorescent than others since
there may be higher concentrations of that specific size of fragment within the sample.

Lastly, it can be observed from the data that although the same amount (10 μL) was added into
wells 2, 4 and 5 in the experiment, the concentration values derived from the absorbances were
different. It is possible to see visual evidence of these differing concentrations by examining the
fluorescence on the gel. While lanes 2 and 4 (containing S1) show concentrated fluorescence at
only one specific spot on the gel (with lane 2 having a very faint fluorescence), lane 5 is
smeared, meaning that this sample may contain excess protein/impurities which result in a higher
absorbance value and thus a higher concentration value. However, when the absorbance ratios
are examined it can be observed that the two samples have similar ratios (1.964
to 1.893), meaning that their purities are close to one another. Therefore, an explanation as to
why lane 2 exhibits such weak fluorescence may be that this well was overloaded and that the
sample therefore diffused, or that the gel was not poured properly, therefore creating
inconsistencies in the migration.

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(7) Interchim. (n.d.). Proteinase K. Www.Interchim.Com.

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