CELL AND MOLECULAR BIOLOGY

LABORATORY

EXERCISE 6
Cheek Cell DNA Extraction
GROUP 2:
BUCTUAN, Riggs B.

ERMINO, Andrea Alexa Joan V.

MORALES, Kaye S.

SANCHEZ, Shellou Grace G.

Experiment date: September 5, 2019

Submission date: September 12, 2019

Submitted to:
 Mr. Paolo Antonio S. Fudalan
INTRODUCTION

The theory behind this experiment is that dead cells continually slough off on both sides
of the body (inner and outer). Recently casted off cells still contain their nucleus and
their DNA genetic material. This DNA can be collected and traced back to its specific
individual.

Detergents dissolve and break down the lipids (fats) and proteins that form the primary
cell membrane and disrupts the bond that hold the membrane together. The cell
contents, including its nucleus, are thus released and become available for further
treatment or isolation. Once cell lysis is completed, you can then proceed with the
removal of the membrane lipids by adding an appropriate detergent or surfactant
followed by the removal of proteins with the aid of proteases. For one, proteases
catalyze the breakdown of contaminating proteins present in the solution to its
component amino acids. It also degrades any nucleases and/or enzymes that may be
present in the sample.

The final step of this experiment requires alcohol. The dissolved DNA will precipitate
when it meets or comes in contact with the alcohol because it is not very soluble in
alcohol. If the procedure is done properly long strands of DNA will form at the interface
where the dissolved DNA and alcohol comes in contact.

In this experiment, it shows us how to isolate human DNA with the use of cheek
cells. A series of reagents and methods will be used to separate the DNA from the cells.
This procedure teaches the properties of cells, cell membranes, and deoxyribonucleic
acid (DNA). The main objectives of the experiment is (1) Learn how to extract DNA from
cells and describe the purpose of the key steps of cell lysis, protein degradation and
DNA precipitation, and (2) Observe the appearance of human DNA.

MATERIALS AND METHODS

Chemicals / Reagents
 Protease and salt solution
 Lysis buffer
 500X Fast Blast DNA stain
Equipment / Glassware

 15mL falcon tubes

 Microcentrifuge tubes
 Micropipettes
 Sterile micropipette tips
 Centrifuge Vortex

PROCEDURE

1. – 2. Gently chew the insides of the mouth (to where the cheek is) for 30 secs and
intake 3 milliliters of water. This is to get the cheek cells off from the side lining or the
walls of your mouth. The water should not be swallowed for the next procedure.

3. We now then add the 2 milliliters of lysis buffer in tube and carefully invert it 5x. Its
purpose is to break open the cells for us to analyze the DNA being extracted from the
nucleus of the cheek cells.

4. After putting lysis buffer into the tube and inverting it five times, the students then
added 5 drops of protease into the solution and inverted the tube five times to let the
solution mix. Protease is a protein enzyme that is added to a mixture to degrade DNA-
associated proteins (DNA are made up of proteins) and other cellular proteins.

5. After the solution was mixed with the protease, the tube was then placed in a water
bath of 50°C for 10 minutes. Heating helps to denature proteins. Like many enzymes,
protease function at optimal temperatures. Checking the time and temperature is very
important because placing the solution for more than the given time and temperature
could cause the DNA to break down.

6. After the water bath, the tube was tilted in a 45° angle and was carefully dropped with
ten milliliters of cold 95% ethyl alcohol. Using ice-cold ethanol increases the yield of
DNA. Low temperatures protect the DNA by slowing down the activity of enzymes that
could break it apart. Cold ethanol helps the DNA to precipitate more quickly.
7. Tube is placed in upright position and is left undisturbed at room temperature for 5
minutes in order to allow the solution to settle thus, preparing it for the next procedure.

8. Tube is then, inverted 5 times in order to allow the DNA to separate and become
more visible which results to its appearance as stringy, white or a more clearer material
out from solution.

9. Liquids surrounding the DNA are then decanted from the container through pipetting
thus, allowing the DNA to remain. The latter is then, transferred to a microcentrifuge
tube and is then placed in a centrifuge machine in order to pull the DNA away from the
particles thus, explaining its visibility and the presence of a supernatant which can be
considered as an excess material that is then removed.

10. A 500 microliter 500X Fast Blast DNA stain is added into the microcentrifuge tube in
order to produce more indication on the DNA. It is then vortexed in order to resuspend
the stain. Tube is then again untouched for at least 10 minutes to let all particles settle.

11-13. As we transfer all the liquid, including the stained DNA to a 15 ml tube containing
ten milliliter mark with fresh 70%. We let it sit for five minutes. We decant as much as of
the ethanol from the tube. Then, we fill the tube to the ten-millimeter mark with 70%
ethanol. We let it sit for another five minutes. In this part, we performed the process of
purification. The initial role of the ethanol and monovalent cations is to remove the
solvation shell surrounding the DNA and permitting the precipitation of the DNA in pellet
form. The ethanol also serves to promote the aggregation of the DNA. With respect to
the washing steps, typically a 70% ethanol solution is used. This permits the
solubilization of the salts whilst minimizing the solubility of the DNA. The salts are
therefore removed due to solubility differences, especially with the aggregated DNA.
The final 100% ethanol wash that is usually employed serves more to permit the easier
evaporation of the ethanol from the DNA pellet in order to prevent any carry over. The
stained precipitated DNA was transfer to helix vial with approximately 500 ml of the
alcohol solution. After the successful DNA extraction, we have now the group's DNA in
a necklace.
GENERALIZATION

Genetic Code or the Blueprint of Life; these terms refer to DNA. All living things,
including animals, plants, and bacteria, have DNA in their cells. DNA is a very long
molecule made up of a chain of nucleotides and the order of these nucleotides is what
makes organisms similar to others of their species and yet different as individuals.
Genes are sections within this long DNA molecule.

The DNA extraction process frees DNA from the cell and then separates it from
cellular fluid and proteins so you are left with pure DNA. The three basic steps of DNA
extraction are 1) lysis (the cell and the nucleus are broken open to release the DNA
inside), 2) precipitation ( separates DNA from this cellular debris using ethanol alcohol)
and 3) purification (rinsed with alcohol to remove any remaining unwanted material and
cellular debris. At this point the purified DNA is usually re-dissolved in water for easy
handling and storage).

CONCLUSION

The extraction of DNA is pivotal to biotechnology. It is the starting point for

numerous applications, ranging from fundamental research to routine diagnostic and
therapeutic decision-making. Extraction and purification are also essential to
determining the unique characteristics of DNA, including its size, shape and function.

Also, the ability to extract DNA is of primary importance to studying the genetic
causes of disease and for the development of diagnostics and drugs. It is also essential
for carrying out forensic science, sequencing genomes, detecting bacteria and viruses
in the environment and for determining paternity.
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APPENDIX
A. Photos

Photo A. The settling DNA of Ms. Morales

 Photo B. Photo Opportunity with our Laboratory Experts who gave us the chance to
experience DNA Extraction of Cheek Cells as our batch will be the last to handle it.

B. Procedures (Lab Manual)