I.

Title Of Experiment
Isolation DNA Of Mouth Ephitelial
II. Date of Experiment
2018, 14th of November 2018 at 09.30-12.00
III. Purpose Of Experiment
Able to do DNA isolation according to procedure with DNA samples taken
from epithelial cells of the mouth.
IV. Basic Theories
a. Deoxyribonuclei Acid (DNA)
DNA, or deoxyribonucleic acid, is the hereditary material in
humans and almost all other organisms. Nearly every cell in a person’s
body has the same DNA. Most DNA is located in the cell nucleus (where
it is called nuclear DNA), but a small amount of DNA can also be found
in the mitochondria (where it is called mitochondrial DNA or mtDNA).
Mitochondria are structures within cells that convert the energy from food
into a form that cells can use (Clark, 1997).
The information in DNA is stored as a code made up of four
chemical bases: adenine (A), guanine (G), cytosine (C), and thymine (T).
Human DNA consists of about 3 billion bases, and more than 99 percent
of those bases are the same in all people. The order, or sequence, of these
bases determines the information available for building and maintaining
an organism, similar to the way in which letters of the alphabet appear in
a certain order to form words and sentences (Hoelzel, 1992).
DNA bases pair up with each other, A with T and C with G, to form
units called base pairs. Each base is also attached to a sugar molecule and
a phosphate molecule. Together, a base, sugar, and phosphate are called a
nucleotide. Nucleotides are arranged in two long strands that form a spiral
called a double helix. The structure of the double helix is somewhat like a
ladder, with the base pairs forming the ladder’s rungs and the sugar and
phosphate molecules forming the vertical sidepieces of the ladder
(Hoelzel, 1992).
An important property of DNA is that it can replicate, or make
copies of itself. Each strand of DNA in the double helix can serve as a
pattern for duplicating the sequence of bases. This is critical when cells
divide because each new cell needs to have an exact copy of the DNA
present in the old cell (Keller & Mark, 1998).
b. Isolation DNA
Deoxyribonucleic acid (DNA) extraction is the process by which
DNA is separated from proteins, membranes, and other cellular material
contained in the cell from which it is recovered. This extraction can be
one of the most labor-intensive parts of DNA analysis. Extraction
methods may require an overnight incubation, may be a protocol that can
be completed in minutes or a couple of hours, or may be a recent
procedure that employs reagents for which this step can be skipped
completely. The DNA extraction process requires careful handling of
biological material to prevent sample contamination and crossover. Tubes
should be carefully labeled, especially when transfers are required. Robots
may be employed to extract reference samples and some evidence
samples, but other evidentiary samples may require the direct attention of
a DNA analyst (Bettelheim & Landesberg, 2007).
The simplest cells, such as bacteria cells, are prokaryotes. These
prokaryotes comprise a lipid bilayer outer membrane and a cytoplasm
containing a circular chromosome, proteins inorganic salts and metal ions,
sugar molecules, and other elements of cell machinery. Humans, animals,
and plants are composed of eukaryotic cells; these cells also have a lipid
bilayer outer membrane and cytoplasm containing proteins, sugars, lipids,
and inorganic ions of various types and function. However, eukaryotic
cells also contain other membrane-enclosed compartments called
organelles. The nucleus of a cell is an organelle that houses 46
chromosomes, and the mitochondria each house a circular DNA
chromosome, all of which direct the production of proteins. The
mitochondrial chromosome is 16,569 bp. The mitochondrial proteins are
used as some of the metabolic machinery for digestion of sugars and fats
and production of most of the energy for the cell. Other organelles are
involved in the synthesis and modification of proteins, sugars, and fats,
and other molecules used by the cell or in its signaling activities. The
genes that code for heritable traits including height, blood type, hair color,
eye color, skin color, and temperament are found on chromosomes in the
nucleus of the eukaryotic cell. In plants, the additional chloroplast
chromosome contains genes for photosynthesis. Forensic scientists are
largely unconcerned with the genes that code for proteins or regulatory
elements of the DNA; however, forensic scientists are interested in
isolating DNA from the nuclear, mitochondria and chloroplast (if present)
chromosomes to evaluate the sequences, base and repeat polymorphisms,
including SNPs and STRs, respectively, that have proved useful for
linking a suspect to a crime scene (Dolpin, 2008).
DNA is highly negatively charged because of its phosphate groups.
It is stabilized by magnesium in the cell when unwound. Nuclear DNA
consists of 3.2 billion bases in humans. It is organized into chromosomes
in part by coiling around positively charged proteins called histones to
form nucleosomes. Magnesium is also integral to the function of
proteases, enzyme proteins that cut up DNA (Holme & Hazel, 1998).
Because of the lipid structure of the cell (and nuclear)
membrane(s), presence of proteases and magnesium, and coiling of DNA
around histones, many of the available DNA extraction procedures have
common elements. Indeed, the extraction of DNA generally follows three
basic steps:
1. Lyse (break open) the cells.
The first stage in DNA isolation is the process of destruction or
destruction of membranes and cell walls. Cell division (lysis) is a
stage from the beginning of DNA isolation which aims to excrete
cells. Cell or tissue destruction stage has several ways, namely by
physical means such as grinding samples using mortar and pestle in
liquid nitrogen or by using the method of freezing-thawing and
irradiation. Another way is to use chemistry and enzymatics. Chemical
destruction such as the use of detergents can dissolve lipids in cell
membranes so that cell membranes destabilize. While enzymatic
methods such as using proteinase K such as to lyse membranes in
blood cells and degrade globular proteins and polypeptide chains in
cell components (Karp, 2008).
In the lysis process using detergents, sodium dodecyl sulphate
(SDS) is often used as the cell membrane dilution stage. Besides
detergent plays a role in cell membrane lyses can also play a role in
reducing the activity of the nuclease enzyme which is a DNA
degrading enzyme. Besides being used SDS, other detergents such as
cetyl trimethylammonium bromide (CTAB) are also often used to lyse
cell membranes in plant DNA isolation. The parameters of success in
the use of CTAB depend on several things. First, the NaCl
concentration must be above 1.0 M to prevent the formation of the
CTAB-DNA complex. Because the amount of water in cell pellets is
difficult to predict, the use of CTAB as a solution solver must be with
NaCl with a minimum concentration of 1.4 M. Second, extracts and
cell solutions containing CTAB must be stored at room temperature
because the CTAB-DNA complex is insoluble in temperatures below
15 ° C . Third, the use of good purity CTAB will determine the purity
of DNA obtained and with very little polysaccharide contamination.
After CTAB was added, the sample was incubated at room
temperature. The purpose of this incubation is to prevent the
deposition of CTAB because CTAB will settle at a temperature of
15°C. Because of its effectiveness in removing polysaccharides,
CTAB is widely used for the purification of DNA in cells containing
many polysaccharides such as those found in plant cells and gram-
negative bacteria such as Pseudomonas, Agrobacterium, and
Rhizobium (Karp, 2008).
 In the use of CTAB buffers other reagents such as NaCl, EDTA,
Tris-HCl, and 2-mercaptoethanol are added. NaCl functions to remove
polysaccharides while 2-mercaptoethanol functions to eliminate the
content of polyphenol compounds in plant cells. 2-mercaptoethanol
can remove polyphenols in plant cells by forming hydrogen bonds
with polyphenol compounds which will then be separated from DNA.
Polyphenol compounds need to be removed in order to obtain good
DNA quality. Polyphenols can also inhibit the reaction of the Taq
polymerase enzyme during amplification. Besides that polyphenols
will reduce the yield of DNA extraction and reduce the level of purity
of DNA. The use of 2-mercaptoethanol by heating can also denaturate
proteins that contaminate DNA.
The concentration and pH of the buffer used must be in the pH
range 5 to 12. The buffer solution with a low pH will cause
depurification and cause the DNA to be distributed to the phenol
phase during the deproteinization process. While the pH of the
solution high above 12 will result in the separation of the double
strand of DNA. The function of buffer solution is to maintain the
structure of DNA during the process of destruction and purification so
as to facilitate removal of proteins and RNA and prevent the activity
of enzymes that degrade DNA and prevent changes in DNA
molecules. To optimize the function of buffer solutions, it takes
concentration, pH, ion strength, and the addition of DNAase inhibitors
and detergents (Karp, 2008).
2. Separate the DNA from the other cell components.
At the DNA extraction stage, chelating agents are often used such
as ethylenediamine tetraacetic acid (EDTA) which acts to inactivate
DNase enzymes that can denaturate DNA isolated, EDTA inactivates
nuclease enzymes by binding to magnesium and calcium ions needed
as DNAse enzyme cofactors. DNA that has been extracted from
within the cell then needs to be separated from the contaminants of
other cell constituent components such as polysaccharides and
proteins so that the DNA obtained has high purity. Phenol is often
 used as denaturing proteins, extraction using phenol causes proteins to
lose their solubility and precipitation which can then be separated
from DNA by centrifugation. Sample after centrifugation 2 separate
phases will be formed, namely the organic phase in the lower layer
and aquoeus (water) phase in the layer while DNA and RNA will be in
the aquoeus phase after centrifugation while denatured proteins will
be at interphase and lipids will be in the organic phase. Besides
phenol, a mixture of phenol and chloroform or a mixture of phenol,
chloroform, and isoamil alcohol can be used to denaturate proteins.
The DNA extract obtained is often also contaminated by RNA so that
RNA can be separated from the DNA extract by means of RNAse
(Keller & Mark, 2009).

Nucleic acids are hydrophilic molecules and are soluble in water.

Besides that, proteins also contain hydrophobic residues which cause
proteins to dissolve in organic solvents. Based on these properties,
there are several deproteinization methods based on the selection of
organic solvents. Usually the organic solvents used are phenol or
chloroform which contain 4% isoamil alcohol. The use of chloroform
isoamil alcohol (CIA) based on differences in the nature of organic
solvents. Chloroform cannot mix with water and its ability to
deproteinize based on the ability of denatured polypeptide chains to
enter or mobilize into the phase between chloroform - water. High
concentrations of protein in the intermediate phase can cause proteins
to precipitation. While lipids and other organic compounds will
separate in the chloroform layer (Keller & Mark, 2009).
3. Isolate the DNA
 After the extraction process, the DNA obtained can be concentrated
through precipitation (separation). In general, ethanol or isopropanol
is used in the precipitation stage. Both of these compounds will
precipitate DNA in the aquoeus phase so that DNA clumps to form a
fiber structure and pellets are formed after centrifugation.
Precipitation also functions to remove chloroform residues from
extraction stages.
The principles of precipitation include the first, reducing the
solubility of nucleic acids in water. This is because water molecules
that are polar surround the DNA molecule in aquoeus solution. The
positive dipole charge of water interacts with the negative charge on
the phosphodiester DNA group. This interaction increases the
solubility of DNA in water. Isopropanol can mix with water, but is less
polar than water. The isopropanol molecule cannot interact with the
polar group of nucleic acids so that isopropanol is a weak solvent for
nucleic acids; second, the addition of isopropanol will remove water
molecules in the DNA solution so that DNA will be precipitated; third,
the use of cold isopropanol will reduce the activity of water molecules
thereby facilitating DNA precipitation.
At this stage of precipitation, precipitated DNA will be separated
from the remaining RNA and protein residues. The residue also
experiences coagulation but does not form a fiber structure and is in
the form of granular precipitate. When the ethanol or isopropanol is
removed and the pellets are dried in the tube, the remaining pellets in
the tube are concentrated DNA. The precipitating process returns with
ethanol or isopropanol before the pellets are dried. DNA purity
isolated. Pellet re-washing which is precipitated by isopropanol using
ethanol aims to remove remaining salt residues. The salts involved in
the extraction process are insoluble in isopropanol so that it can be
precipitated with DNA, therefore it is necessary to re-precipitation
with ethanol after precipitation with isopropanol to remove salt
residue.
 After the precipitation process was carried out and ethanol was
washed, ethanol was then removed and the pellets dried, the treatment
was aimed at removing ethanol residues from DNA pellets. The
removal of ethanol residues was carried out by evaporation because
ethanol easily evaporated. In the washing stage, ethanol is usually
mixed with ammonium acetate which aims to help separate unwanted
contaminants such as dNTP and oligosaccharides that are bound to
nucleic acids (Keller & Mark, 2009).
V. Tools And Materials
A. Tools
1. Centrifuge 1 piece
2. Digital Scales 1 piece
3. Microcentrifus tube 3 pieces
4. Beaker Glass 3 pieces
5. Test tube rack 1 piece
6. Pipette 3 pieces
7. Test tube 3 pieces
8. Vortex 1 piece
9. Micropipette 1 piece
10. Pipete volume 1 piece
B. Materials
1. Isotonic solution 10 mL
2. Aquades 10 mL
3. Mineral water 10 mL
4. Buffer tris EDTA 1 mL
5. NaCl 2,5 M solution 100
6. Cool ethanol 1 mL
VI. Lanes Work
1. Collecting of Cells
± 10 mL Isotonic ± 10 mL ± 10 mL Mineral
Solution Aquadest Water

Used to rinse for about 1 minute

Entered into 3 different beaker glass

Sample Sample Sample

Solution Solution Solution

2. Lisis Cells and Protein Digestion

1,5 mL Sample Solution

Taken with volumetric pipette

Entered into micro centrifuge tube
Centrifuge with 10.000 rpm in
microcentrifuge for 1 minute
Separated by decantation process

Residue Filtrate

Added 1,5 mL sample solution

Centrifuge at 10.000 rpm until 1
minute
Separated
Repeated until 2 times

Residue Filtrate

Added 1 mL buffer tris-EDTA

Vortex microcentrifuge tube until 1
minute
Observed the result

Lysis Cell solution

3. Precipitation of DNA
Lysis cell solution in
microcentrifuge tube

Added 100 µL NaCl 2,5 M

Mixtured
Mised in another test tube carefully
Added 1 mL cool ethanol
Let in until 5 minutes

DNA dots between buffer and ethanol

boundaries (drifts likes white thread)
 VII. Observation Result

Experiment Result
No. Procedure Prediction/Reaction Conclusion
Before After
1 Collecting of Cells  Isotonic  Isotonic sample solution: Turbidiry of solution Turbidiry based on
solution: turbid solution contain DNA experiment is
 Aquadest sample solution:
turbid Isotonic solution >
turbid solution
solution mineral > aquadest
 Mineral water sample
 Aquadest:
solution: turbid solution
colorless
solution
 Mineal water:
colorles
solution
2 Lisis Cells and Protein Digestion  Aquadest  Isotonic sample solution +
sample centrifuge = there is white
solution: precipitate and colorless
 Separated:
turbid
Residue: white precipitate
 Isotonic
Filtrate: colorless
sample  +sample: turbid
 +centrifuge: white
solution:
precipitate and colorless
turbid
 Residue + sample: turbid
 Mineral water
 +centrifuge: white
sample precipitate and colorless
 Separated:
solution:
Residue: white precipitate
turbid Filtare: colorless solution
 +buffer tris EDTA: turbid
 +vortex: turbid
 Aqudest sample solution
+ centrifuge: there is white
precipitate
 Separated:
Residue: white precipitate
Filtare: colorless solution
 Residue + sample: turbid
 +centrifuge: white
precipitate and colorless
 Residue + sample: turbid
 +centrifuge: white
precipiatte and colorless
 +buffer tris EDTA: turbid
 +vortex: turbid

 Mineral water sample

solution + centrifuge:
white precipitate and
colorless
 Separated:
 Residue: white precipitate
Filtare: colorless solution
 Residue + sample: turbid
 +centrifuge: white
precipitate and colorless
 Residue + sample: turbid
 +centrifuge: white
precipiatte and colorless
 +buffer tris EDTA: turbid
 +vortex: turbid
3 Precipitation of DNA  Lysis cell: Isotonic Solution DNA in Isotonic > Based on the
turbid  Lysis + NaCl: colorless mineral water > experiment, we
solution isotonic cell aqudest cannot state a
 NaCl:  +ethanol: colorless
conclution because
 Let in for 5 minutes: DNA
colorless
DNA is not isolated.
is not isolated
solution
 Cool ethanol:
Mineral Water
colorless
 Lysis + NaCl: colorless
solution
mineral water
 +ethanol colorless
 Let in for 5 minutes: DNA
is not isolated
Aquadest
 Lysis + NaCl: colorless
 mineral water
 +ethanol colorless
 Let in for 5 minutes: DNA
is not isolated
VIII. Analysis and Explanation
DNA is abbreviation of deoxyribonucleic acid, organic chemical of complex
molecular structure that is found in all prokaryotic and eukaryotic cells and in many
viruses. DNA is a double-helix polymer, a spiral consisting of two DNA strands
wound around each other. The breakthrough led to significant advances in
scientists’ understanding of DNA replication and hereditary control of cellular
activities.

Each strand of a DNA molecule is composed of a long chain of monomer

nucleotides. The nucleotides of DNA consist of a deoxyribose sugar molecule to
which is attached a phosphate group and nucleoside. Nucleoside consists of ribose
and one of four nitrogenous bases: two purines (adenine and guanine) and two
pyrimidines (cytosine and thymine). The nucleotides are joined together by
covalent bonds between the phosphate of one nucleotide and the sugar of the next,
forming a phosphate-sugar backbone from which the nitrogenous bases protrude.
One strand is held to another by hydrogen bonds between the bases; the sequencing
of this bonding is specific such as adenine bonds only with thymine, and cytosine
only with guanine.
DNA replicates by separating into two single strands, each of which serves as
a template for a new strand. The new strands are copied by the same principle of
hydrogen-bond pairing between bases that exists in the double helix. Two new
double-stranded molecules of DNA are produced, each containing one of the
original strands and one new strand. This semiconservative replication is the key to
the stable inheritance of genetic traits.
 Within a cell, DNA is organized into dense protein-DNA complexes called
chromosomes. In eukaryotes, the chromosomes are located in the nucleus, although
DNA also is found in mitochondria and chloroplasts. In prokaryotes, which do not
have a membrane-bound nucleus, the DNA is found as a single circular
chromosome in the cytoplasm. Some prokaryotes, such as bacteria, and a few
eukaryotes have extrachromosomal DNA known as plasmids, which are
autonomous, self-replicating genetic material. Plasmids have been used extensively
in recombinant DNA technology to study gene expression.
DNA has two main biological functions. DNA mainly functions to store /
determine the biological characteristics of every living thing according to the
regulation of very specific molecular connections. Second, DNA serves the purpose
of biological synthesis in terms of the creation of cellular proteins and RNA
molecules. DNA functions can be mentioned:
1. Carrier of DNA genetic information
2. Acting in self-duplication and inheritance
3. Expression of genetic information
4. DNA directs RNA synthesis under a chemical process known as
transcription
5. As a code for how to activate proteins or deactivate genes
6. Make protein by synthesis process
7. As autocatalyze and heterocatalyze

In the experiment of isolation DNA of mouth ephitelial is used to do DNA

isolation according to procedure with DNA samples taken from epithelial cells of
the mouth. DNA isolation is carried out in order to separate DNA from other
materials such as protein, fat, and carbohydrates. There are three main principles in
DNA isolation, namely destruction (lysis), extraction or separation of DNA from
solid materials such as cellulose and proteins, and DNA purification. Some things
that need to be considered in the process of DNA isolation include having to
produce DNA in the absence of contaminants such as proteins and RNA, the
method must be effective and can be done for all species of methods that cannot
change the structure and function of DNA molecules, and the method must be
simple and fast.

a. Collecting of cells
 The first procedure in the lab was to obtain epithelial mouth cell samples
using several solvents including aquades, isotonic water and mineral water.
Take 10 ml of solvent for each solvent. place on different beaker glaases. use to
rinse for 10 minutes for each solvent. The entire solution at first is colorless
solution and turns into a litle turbit solution after it is used to rinse the mouth.
The gargling process functions to obtain oral epithelial cells that will be isolated
to obtain DNA.
Isotonic solutions contain NaCl salts which are used to maintain the
structure of DNA molecules in cells and facilitate deproteinization. Therefore,
in isotonic solvents it allows the bond to sell more than other solvents.
b. Lisis cells and protein digestion
The first stage in DNA isolation is the process of destruction or
destruction of membranes and cell walls. Cell division (lysis) is a stage from the
beginning of DNA isolation which aims to excrete cells. First, take the turbit
solution of sample using volumetric pipette to get an accurate sample volume.
Enter in the microcentrifuge tube and use a centrifuge at 10,000 rpm for 1
minute. After that, do decantation to remove the part of the palette and leave a
part of the supenatant containing DNA. Centrifuge speed that is too high or too
long can cause cells to lyse faster and DNA out of the cell after that is wasted
during the decantation process to separate the palette and supernatant. Do this
procedure with three repetitions to get the expected DNA.
Second, enter 1 ml of tris-EDTA as buffer solution after the third
decantation process. Vortex the micocentrifuge for 1 minute. The concentration
and pH of the buffer used must be in the pH range 5 to 12. The buffer solution
with a low pH will cause depurification and cause the DNA to be distributed to
the phenol phase during the deproteinization process. While the pH of the
solution high above 12 will result in the separation of the double strand of
DNA. The function of buffer solution is to maintain the structure of DNA
during the process of destruction and purification so as to facilitate removal of
proteins and prevent the activity of enzymes that degrade DNA and prevent
changes in DNA molecules. Tris-EDTA which acts to inactivate DNase
enzymes that can denaturate DNA isolated, EDTA inactivates nuclease enzymes
 by binding to magnesium (Mg2+) and calcium ions which are needed as DNAse
enzyme cofactors to puncture the cell wall so that DNA can come out at a later
step. Use of vortex to help widen the torn cell wall.
Mineral water as solvent in sample, the turbit solution after centrifuge
became two layers (colorless in upper and turbid in below) and throw away the
upper as palette and save the below as supernatant. After added colorless
solution of buffer tris-EDTA became colorless solution. It has the same
condition in aquades as solvent system and isotonic as solvent system.
c. Precipitation of DNA
After the process of separating DNA from cells, NaCl is added to 100
micoliters to remove contaminants such as carbohydrates and proteins. Taking
the solution using a micropipette to produce accurate volumes. After that
colorless solution of 1 ml of ethanol 70% is added. Ethanol will precipitate
DNA in the aquoeus phase so that DNA clots to form a fiber structure.
The three samples possessed: DNA in mineral water, isotonic solution
and aquades produce a colorless solution. Thus, observing the formation of
fiber DNA requires additional light to facilitate observation. From the
observations of three samples in different solvents, there was no DNA fiber
which meant that DNA was not isolated during the process.
IX. Discussion
Based on the practicum, there is no isolated DNA fiber. This can be due to several
processes including:
1. Cells obtained when gargling too little.
2. DNA is also wasted during the separation of the palette and supernatant in the
cell lysis stage, this is due to the color similarity between the sediment that
wants to be stored and the solution that is removed.
3. Ethanol added has not reacted to agglomerate DNA.
X. Conclusion
Based on the experiment can be concluded that DNA is not isolated because there
was no fiber DNA that can be observed.
XI. References

Bettelheim & Landesberg. 2007. Laboratory experiments for general organic and
biochemistry. New Delhi : Oxford University Press.
Clark, Melody S. 1997. Plant Molecular Biology : A laboratory manual. New
Delhi : Tata McGraw-Hill Publishing Company Ltd.
Dolphin, W. D. 2008. Biological Investigations. Delhi : UBS Publisher’s
Distributors Ltd
Hoelzel, A. R. 1992. Molecular Genetic Analysis of Populations. London:
Macmillan Press Ltd.
Holme, D. J. & Hazel P. 1998. Analytical Biochemistry. Delhi : CBS Publishers &
Distributors.
Karp, Gerald. 2008. Cell and Molecular Biology. Delhi : CBS Publishers &
Distributors.
Keller, G. H. & Mark M. M. 2009. DNA probes. New Delhi : Tata McGraw-Hill
Publishing Company Ltd.
Tim Dosen. 2018. Panduan Praktikum Biokimia. Surabaya: Jurusan Kimia Unesa.
 ATTACHMENT
ANSWER OF QUESTION

1. Draw the structure of DNA!

2. How to determine the amount of DNA from the isolation using a

spectrophotometer?
To test quantitative and qualitative DNA isolation, measurements can be made
using UV-Vis spectrophotometry and agarose gel electrophoresis. Quantitative
DNA testing with UV-Vis spectrophotometry, pure DNA can absorb ultraviolet light
due to the presence of purine and pyrimidine bases. Double DNA bands can absorb
UV light at  260 nm, while protein or phenol contaminants will absorb light at 
280 nm. So that the purity of DNA can be measured by calculating the absorbance
value of  260 nm divided by the absorbance value  280 (Å260 / Å280), and the
DNA purity value ranges from 1.8-2.0. As well as to measure DNA concentration
the following formula is used:

[DNA] = Å260 x 50 x dilution factor

Å260 = Absorbance value at  260 nm

[DNA] = Å260 x 50 x faktor pengenceran

50 = solution with an absorbance value of 1.0 equal to 50 ug of double strand of
DNA per mL

The standard method used for identification, separation and purification of DNA
fragments is using agarose gel electrophoresis. Electrophoresis migration of DNA
through agarose gel is influenced by the size and conformation factors of DNA
molecules, agarose concentration, electric current and temperature. Dyes of
Ethidium Bromide (EtBr) are used to identify and measure semi-qualitative DNA
fragments fragmented in a gel. This EtBr will be bound between two double strands
of DNA, so that the DNA band / band in the agarose gel will glow, because this dye
contains fluorescence. EtBr can be given to each sample that will be inserted into
the gel well or mixed into agarose gel before the gel is printed in the gel mold.
Fluorescence intensity can be measured using standard DNA markers, so the DNA
quantity can be estimated, for example between 0.50 to 20 ug / mL.
PICTURES

PREPARATION

Tools: volumetric flask, propipette, Materials: mineral water, isotonic

beaker glass, rack, test tube, water, aquades
measurement flask, pipette

Tools: vortex Tools: centrifuge

COLLECTING OF CELLS

10 ml of each solvent Sample solution

LISIS CELLS AND PROTEIN DIGESTION
Isotonic sample in microcentrifuge Aquades sample in microcentrifuge

Mineral sample in microcentrifuge Sample in microcentrifuge

Sample after had treatment in decantation

microcentrifuge

Sample in vortex Added 1 ml of buffer tris EDTA

Moved sample in different test tube
PRECIPITATION OF DNA

Added 100 microliters NaCl 2,5M Added cool ethanol

Find the DNA by using lamp