UPD-IC | Chem 145.

1 | Cervera: Extraction, Purification and Characterization of Genomic DNA from a Microbial Culture Page | 1
Extraction, Purification and Characterization of Genomic DNA from a Microbial Culture
S.L.M. Cervera*, Z. Escobido, C. Frivaldo, S. Reyes
The main objectives of the experiment are to successfully extract, purify, quantify and characterize the genomic
DNA of cultured microbes obtained from a watch. The microbial culture was incubated for3 weeks and upon
checking, maggots have contaminated the agar sample. No significant colonies were observed but it was yellowish
white with pungent odor. Threadlike fibers were obtained after the extraction using an extraction kit It was
subjected to Agarose Gel Electrophoresis (AGE) and after viewing the gel in UV light, no observable bands were
formed indicating very minimal to no presence of DNA.

Introduction various locations is critical in clinical studies, food and

Deoxyribonucleic acid or DNA is a polymer made
of nucleotides that provide the chemical basis for
inheritable characteristics of all cellular organisms. A
nucleotide (figure 1) is composed of the following: a
nitrogenous base, a deoxyribose and a phosphate
group.
water industries. Before identification and analysis,
microbe samples are isolated. The methods involve
disruption and lysis of the source followed by protein
and contaminant removal in the DNA sample. Organic
extraction, salting out methods, and filter paper-based
methods are some of the examples of extraction
techniques2.
To extract, purify, and characterize DNA samples
from starting materials, addition of organic solvents and
centrifugation must be performed to extract and purify
the genomic DNA from a microbial culture, followed
characterization using AGE

Methodology

Preparation of Microbial Culture

Figure 1. Structure of nucleotide
The genetic information stored in the DNA is
defined by the sequence of bases. There are two
types, the pyridines and purines. The pyrimidines are
cytosine and thymine and the purines are guanine and
adenine. The acceptors and donors of each base in the
strand are connected by appropriately positioned
hydrogen bonds. This makes sure that A pairs with T
and G pairs with C. The DNA in the cell usually
adopts a double‐stranded helical form, with
complementary base pairing holding the two strands
together.1
Figure 2. Double helix structure of DNA
The detection and analysis of microorganisms found on
Lysogeny agar broth was heated in a water bath at
temperatures between 80 and 900C until the entire
agar was liquid. It was cooled down to 40-500C then
slowly poured into the petri dish to cover half of the
bottom of the dish. It was covered immediately to
avoid contamination and was tilted back and forth to
entirely cover the bottom of the dish. It was allowed
to stand in room temperature for the agar to solidify.
Growing Bacteria
A clean cotton swab was dampened with distilled
water and rolled on he surface of the watch. The lid
of the ish was slightly opened to give way for the
swab. A squiggly lined was drawn on the surface of
the agar. The petri dish was sealed with the parafilm
and it was placed inside the locker. Changes were
analyzed the following session.
Extraction of Genomic DNA
The cells were scraped from the surface of the agar
plate and were put in a microcentrifuge tube. The
tube was centrifuged and then the liquid broth was
decanted afterwards. The pellets were resuspended
in 1 mL of 0.05 M Tris-EDTA buffer pH 8.0 and
shaken until pellets dissolved. A hundred microliters
of 10% SDS was slowly added to the solution. It was
heated in a hot water bath at 600C for 20 minutes.
After removing the tube from the water, a Pasteur
UPD-IC | Chem 145.1 | Cervera: Extraction, Purification and Characterization of Genomic DNA from a Microbial Culture
pipette was filled with cold isoamyl alcohol and was
realesed into the solution. Let it stand for 2-3
minutes. White, fibrous precipitate was observed
precipitating in the alcohol-water interface and it was
centrifuged after to pellet out the DNA. The alcohol
was decanted and the DNA pellet was air-dried after.
The DNA was immediately resuspended using 1 .5
mL 0.05 M Tris-EDTA buffer pH 8.0.
Determination of Nucleic Acid Concentration and
Purity
Five hundred microliters of DNA solution was
pipette out and diluted to 2.0 mL using 0.05 M Tris-
EDTA buffer pH 8.0. The absrbance of the solution
was measured at 260 and 280 nm using the 0.05 M
Tris-EDTA buffer pH 8.0 as blank.
Gel Preparation
An amount of 0.175 grams gel powder was dissolved
in 25 mL of 1X TAE buffer. The agarose mixture
was heated with occasional stirring. Th moten
agarose was allowed to cool to 370C. Three hundred
microliters of GelRed was added and swirled to mix.
The agarose solution was poured gently onto the gel
tray and the comb was placed over the gel. The gel
was allowed to solidify for 20 to 30 minutes at room
temperature.The comb was carefully removed and the
wells were flushed with buffer before loading the
sample.
Sample Preparation and Loading
Three small square pieces of parafilm was cut—one
for the low moceular weight ladder, one for the high
molecular weight lader and the other for the DNA
extract. Ten microliters of loading buffer was pipette
out onto each of the parafilm. Twenty microliters of
the DNA sample was added to the loading buffer.
The spot was drawn up and down to mix and 20 µL
of the mixture was loaded on the well.
Running the Gel
The gel chamber was filled with running buffer. Te
power supply was set and maintained to 40-60 V.
The apparatus was turned off once the trackng dye
reaches 80% of the gel length. Total run time was
about 30 to 45 minutes.
Viewing the Gel
The gel was carefully removed from the AGE
setup and was placed in a transparent flat-bottomed
container. The gel was visualized by placing it on a
UV Trans-illuminator immediately after the
electrophoretic run. The gel was suspended in 1X
TAE and stored at 40C for future use.
Page | 2
Results and Discussion
The sample was collected from the surface of a
wrist watch. Due to the extended length of bacteria
culture growth in the agar plate, it was contaminated
by presence of maggots as seen in Figure 1 in the
appendix. The isolated sample was yellowish in color
and has very pungent odor.
DNA appears to be threadlike and fibrous because
they are packed into thread-like structures which are
called chromosomes. They are made up of DNA
strands that are tightly coiled around proteins called
histones. If the extract obtained appears fragmented
and powdery instead of threadlike, this means that the
one obtained is plasmid DNA and not genomic DNA.
Centrifugation was used to separate the bacteria
cells from other cell debris and substances as well as
lyse the cell. Lysing the cell breaks down the cell
wall of the cell releasing the DNA.10% SDS (sodium
dodecyl sulfate) was added because it is a strong
anionic detergent that could solubilize the lipids and
proteins forming the membranes. It removes the
negative ions from the proteins and deforms its
conformation which then destroys its structure. This
encourages the breaking down of the nuclear
envelopes which then expose the chromosomes
holding the DNA. SDS also helps in the release of
DNA from the histones and other binding proteins by
denaturation.
The primary function of Proteinase K is to
inactivate nucleases; they leave the molecules whole
and intact. Proteinase K also unwinds DNA by
degrading histone proteins in which chromosomes
coil around. Removal of histones linearizes the DNA
for downstream functional assays.
Isoamyl alcohol is an anti foaming agent and it
stabilizes the interphase (coagulated proteins). It also
helps in the precipitation of proteins and
carbohydrates. The addition of isoamyl alcohol
produced 2 layers because of the solubility. The DNA
is more soluble in the aqueous portion of the organic–
aqueous mixture.
Solid DNA extract was resuspended in Tris-EDTA
buffer pH 8.0 because EDTA binds divalent cations
such as calcium and magnesium. These ions help
maintain the integrity of the cell membrane and since
EDTA eliminates these ions, it destabilizes the
membrane. Tris, the main buffering component,
maintains the pH of the buffer at a stable point which
is usually at pH 8.0.
The general formula to get the number of double
stranded DNA pieces is 2n. The number increases by
powers of 2 in each cycle, so that after n cycles there
UPD-IC | Chem 145.1 | Cervera: Extraction, Purification and Characterization of Genomic DNA from a Microbial Culture Page | 3

are 2n numbers of copies. Using the formula, 10 [2] Bowien, B.; Durre, P. Nucleic Acids Isolation
cycles will give 1,024 copies and so on. Methods. American Scientific Publishers: America,
2003;.
In the preparation of the gel, as seen in appendix 1,
[3]Bowater, Richard P, and Waller, Zoë AE(Apr
only 1 band was visible after procedure. Several
2014) DNA Structure. In: eLS. John Wiley & Sons
factors affect the migration such as: DNA/RNA
Ltd, Chichester. http://www.els.net [doi:
molecular weight, voltage, agarose, buffer, and
10.1002/9780470015902.a0006002.pub2]Appendix
visualization. Gel electrophoresis makes use of
[4] Kennedy, Suzanne (2010) How Extraction Kits
components such as agarose, TAE, and .
Work in the Lab
Ethidium bromide (EtBr) was used as a fluorescence dye [5] Elkins, Kelly M. (2012, August 10) DNA
for AGE. This dye binds to DNA through intercalation. Extraction https://doi.org/10.1016/B978-0-12-
EtBr intercalates to both RNA and DNA. RNA is one of 394585-3.00004-3
the contaminating proteins present, and thus an addition of [6] Wright, M. H., Adelskov, J., & Greene, A. C.
ribonuclease must again be done to completely denature the
(2017). Bacterial DNA Extraction Using Individual
RNA which leads to the decrease in amount of fluorescence
detected with respect to the amount of RNA. Enzymes and Phenol/Chloroform Separation. Journal
of microbiology & biology education, 18(2), 18.2.48.
Spin columns contain a silica resin that selectively binds
doi:10.1128/jmbe.v18i2.1348
DNA (or RNA), depending on salt conditions and other
factors influenced by the extraction method. For the [7] Butler, John M. (2012) DNA Extraction
visualization of data, GelRed is known to be safer and Methods—Organic (Phenol-chloroform) Extraction
more stable than ethidium bromide.
Extraction kits are used to extract RNA or DNA. In
using these kits, it undergoes 5 important steps;
namely, cell lysis, purification, washing, dry spin,
and elution. In cell lysis, the buffers used have high
concentrations of chaotropic salts. These salts
destabilize hydrogen bonds, VDW forces, and
hydrophobic interactions which lead to
destabilization of proteins and nucleases. Afterwards,
the addition of lasohol will further enhance and
influence the binding of nucleic acids to silica. After
centrifuging the lysate through a silica membrane, the
desired nucleic acids will be bound to the column.
Subsequent addition of Tris buffer will hydrate the
nucleic acids which facilitates elution. The final step
involves the release of the DNA from the silica.
Conclusion
Based on the data gathered and analyzed, it is
evident that the experiment was successful in
extracting threadlike fibers but due to the extended
incubation, the agar plate was contaminated and
therefore compromised the experiment. After
viewing the gel using UV light, it was seen that no
bands were formed. This means that very minimal or
no DNA was extracted. It is recommended to follow
a shorter incubation time and seal the plate properly
to avoid contamination.

References

[1] Alex, Dr.. (2018, April 23). What Is the Function

of a Tris Buffer in DNA Extraction? Sciencing.
Retrieved from https://sciencing.com/function-tris-
buffer-dna-extraction-6370973.html
UPD-IC | Chem 145.1 | Cervera: Extraction, Purification and Characterization of Genomic DNA from a Microbial Culture Page | 4

Appendix

Figure 1. Agar plate after three weeks of incubation

Figure 2. Gel under UV Light

UPD-IC | Chem 145.1 | Cervera: Extraction, Purification and Characterization of Genomic DNA from a Microbial Culture Page | 5