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- Use a sterile cytobrush and insert into mouth.
- Brush the cytobrush on the inside of your cheek 25 times.
- Swirl the cytobrush in 100 μl of Chelex suspension (10% w/v).
- Place a centrifuge tube with Chelex and cell suspension on a 100 °C heat block for 10 minutes.
- Centrifuge the tubes at maximum speed for 5 minutes.
- DNA is in the supernatant. Avoid beads at the bottom.
- Store DNA at -20 °C.
PCR with PCR Beads
- Add 22 μl of the primer mix (forward and reverse) to beads.
- Ensure that the bead is dissolved.
- Add 3 μl of DNA.
8.7: Cheek Cell DNA Extraction - Biology
DNA Extraction from Cheek Cells
adapted by Roxane Bonner
Laboratory of Molecular Systematics and Evolution University of Arizona
1. Vigorously swish 15 mL of Gatorade in your mouth for 30 seconds.
2. Chew on your cheeks while swishing so you get as many cells as possible.
3. Remember: more cells = more DNA. Spit the Gatorade back into your cup, then pour it into a new 15 mL plastic tube.
4. Mark your tube and lid with your initials. Find a partner and make sure your tubes each have the same volume.
5.Add more Gatorade if necessary to make the volumes equal. Place your tubes opposite each other in a centrifuge.
6. Spin the tubes on high for 3-5 minutes to collect cells at the bottom of the tube.
7. Pour the liquid into a waste container. Repeat steps #1-4.
8. Pour the Gatorade/Spit from your second mouthwash into the tube with the cells from your first mouthwash.
9. Add 2.0 mL of Lysis Buffer to the cell pellet. Mix by carefully flicking the tube. OPTIONAL .
10. Add 50 (l of Proteinase K (see teacher). Flick the tube to break up the cell pellet.
11. Incubate the cells at 65-70 C for atleast one hour, preferably overnight. Before proceeding, the solution should be clear and the cells should not be visible.
12. Add 1.0 mL of 5M NaCl. Mix well, then centrifuge for 10 minutes on high speed.
13.Transfer the liquid into a new 15 mL tube. Do not let the stuff on the bottom fall into the new tube. If necessary, use a clean pipet to transfer only the liquid.
14. Add 4 mL of cold 95% ethanol to the liquid in the new tube.
15. Mix well by rocking the tube gently back and forth until the DNA becomes visible. It should look like fine white fibers or lint.
DNA Extraction from Cheek Cells Directions:
Answer all questions in complete sentences.
1. How did we collect the cell pellets from the mouthwash solution?
2. Why do some people have larger cell pellets than others?
3. Why don't we see a cell pellet when we remove our tubes from the water bath?
4. What was the purpose of each of the following components?
- Lysis Buffer & Detergent
- Water Bath & Heat
- Sodium Chloride (NaCl)
5. Show where the fats, carbohydrates, proteins and DNA are in each of the following stages.
A cytobrush is a small brush that is traditionally used for performing pap tests. These are sterile and convenient for single usage inside the cheek of the mouth. Brushes are swirled in a solution of Chelex beads so that the chelating agents in the solution neutralize the action of divalent cations to protect the DNA . Placing these tubes of cells and Chelex onto a boiling heat block is designed to sterilize the solution for safe working conditions. An alternative to this use of Chelex is to displace the cells into a lysis buffer that will burst the cells and protect the DNA.
- Use sterile cytobrush and insert into mouth
- brush cytobrush on inside of cheek 25 times
- Swirl cytobrush in 100 &mul of Chelex suspension (10% w/v) or lysis buffer containing EDTA
- Place centrifuge tube with Chelex and cell suspension on 100 °C heat block for 10 minutes
- Centrifuge tubes at maximum speed for 5 minutes
- DNA is in the supernatant. (avoid beads at bottom)
- Store DNA in -20 °C
2. Precipitation of DNA
After the cell and nuclear membranes are broken down, the lipid molecules must be removed. A lab technician adds a highly concentrated salt solution. This causes the detergent and other cellular debris, such as proteins, to precipitate. To precipitate means to form a solid that separates from the solution.
The DNA remains dissolved in the liquid solution. It can be removed from the cellular debris by centrifugation. In centrifugation, the liquid solution spins at high speed so that the precipitate collects as a pellet at the bottom of a tube. The DNA, which is still dissolved in the liquid, can be moved to a new sample tube. Alternatively, the precipitate can be filtered out of the solution. That way, the liquid containing the DNA is left behind .
DNA in the nucleus is wrapped around proteins called histones. This helps organize the DNA into chromosomes. To remove the histone proteins, a protease can be added. A protease is an enzyme that breaks down proteins.
Extraction of DNA from Cheek Cells
Extraction of DNA from Cheek Cells
February 29th 2013
DNA, deoxyribonucleic acid, is the genetic material of every living organism and is found in the nucleus of eukaryotic cells. DNA is often called the ‘blueprint for life’ because it contains the necessary information to carry out all the living processes of the cell (1).The purpose of this lab was to extract DNA from human cheek cells. The isolated DNA could be used inmapping or sequencing, PCR, crime scene investigation orother downstream applications(2). MATERIALS AND METHODS
To extract DNA from cheek cells, 3 mL of water was added to a labeled, 15 mL tube. The inner lining of the cheek was then chewed for approximately 30 seconds. The 3 mL of water was used to rinse the mouth for 30 seconds, after which the water and the cheek cells were expelled back into the 15 mL tube. Using a plastic transfer pipet, 2 mL of lysis buffer was added to the tube containing the water with the cheek cells. The tube was then capped and gently inverted 5 times to lyse the cells. Five drops of protease and salt solution were added to the sample. The tube was capped and inverted 5 times. The sample was incubated in a water bath at 50°C for 10 minutes. Following incubation, 10 mL of cold ethanol was slowly added while holding the tube at a 45° angle. The tube was placed upright at room temperature for five minutes, after which the sample was inverted 5 times. The DNA extraction protocol and all materials and reagents were provided by Bio-Rad(2). RESULTS
Throughout the experiment, numerous observations were made.The water containing cheek cells was white or cloudy in appearance. Upon addition of the lysis buffer and protease, the solution became clear. A white precipitate was visible following the addition of cold ethanol. DISCUSSION
Extraction of DNA from cells is a relatively straight-forward and simple process. It is routinely carried out in laboratories and has numerous downstream applications.
What You Need for DNA Extraction:
What You Do:
1. Create a saline solution in a beaker by adding two lab scoops of salt to approximately 25 ml of distilled water. Stir until the salt is completely dissolved.
2. Pour the saltwater into the paper cup.
3. Without swallowing, drink a mouthful of the solution from the paper cup and swish it back and forth for at least 30 seconds, occasionally scraping your teeth along the inside of your cheeks as you do. It’s best to do this with a clean mouth, i.e. not right after lunch.
4. Spit your mouthwash solution back into the cup. Then bend the cup into a sort of spout and pour the mouthwashed solution into the test tube until it fills about one-half inch of the bottom of the test tube.
5. Carefully add two drops of the liquid soap.
6. Tilting the test tube approximately 45 degrees, use the pipette (or dropper on the alcohol bottle) to add 20 drops of the chilled alcohol so it slides down the test tube without disturbing the solution. Since it’s less dense, the alcohol will sit atop the mouthwash and soap solution.
7. Tightly put the cap on the test tube and very slowly and gently tilt it upside down then right side up three times. Do it carefully so as not to make bubbles.
8. Let the test tube sit undisturbed in an upright position for one minute. At this point, you should begin to see a milky white thread, possibly interspersed with bubbles, appear between the solution and the alcohol. That’s your DNA! After several minutes, the DNA should be suspended in the alcohol layer.
9. If you wish, insert your skewer or stir rod into the test tube and gently wind the DNA around it.
10. To save it, carefully scrape it into the small vial with a few drops of alcohol. Stored in the freezer, you can preserve your DNA almost indefinitely!
Step 1. Breaking cells open to release the DNA
The cells in a sample are separated from each other, often by a physical means such as grinding or vortexing , and put into a solution containing salt. The positively charged sodium ions in the salt help protect the negatively charged phosphate groups that run along the backbone of the DNA.
A detergent is then added. The detergent breaks down the lipids in the cell membrane and nuclei . DNA is released as these membranes are disrupted.
Step 2. Separating DNA from proteins and other cellular debris
To get a clean sample of DNA, it’s necessary to remove as much of the cellular debris as possible. This can be done by a variety of methods. Often a protease ( protein enzyme) is added to degrade DNA-associated proteins and other cellular proteins. Alternatively, some of the cellular debris can be removed by filtering the sample.
Step 3. Precipitating the DNA with an alcohol
Finally, ice-cold alcohol (either ethanol or isopropanol ) is carefully added to the DNA sample. DNA is soluble in water but insoluble in the presence of salt and alcohol. By gently stirring the alcohol layer with a sterile pipette, a precipitate becomes visible and can be spooled out. If there is lots of DNA, you may see a stringy, white precipitate.
Step 4. Cleaning the DNA
The DNA sample can now be further purified (cleaned). It is then resuspended in a slightly alkaline buffer and ready to use.
Step 5. Confirming the presence and quality of the DNA
For further lab work, it is important to know the concentration and quality of the DNA.
Optical density readings taken by a spectrophotometer can be used to determine the concentration and purity of DNA in a sample. Alternatively, gel electrophoresis can be used to show the presence of DNA in your sample and give an indication of its quality.