The purpose of this experiment was to determine what factors cause degradation in Oncorhynchus sp. DNA. The factors were exposure to a magnetic field with an oscillating polarity, cooling and heating the solution, and exposure to high-frequency sound waves.

There were two phases to this experiment. Similar procedures were used for each. In Phase 1, I had only one manipulated variable. I placed samples of the DNA solution on a magnetic stirrer for several different time intervals. I did this along with other follow-up tests, involving a SonikatorÒ . After each test, I spooled the DNA from the solution, and used a spectrophotometer to measure the absorbency of the DNA fragments. Then, I calculated the percentage of DNA remaining in the solution.

For Phase 2, the same procedures were used, except this time I tested the DNA against different factors. I still used the magnetic stirrer, but also a high-frequency SonikatorÒ , and I cooled a separate DNA solution in a cold-store room at about 4° C. (Both solutions came from the same stock DNA solution) Then, I slowly brought the mixture to room temperature by placing it in a sink full of water at 22° C. I then spooled and analyzed the remaining DNA solution for degradation.

The results of Phase 1 were intriguing. My hypothesis had been correct, but I don't think it was for the right reasons. The magnetic field with an oscillating polarity did in fact cause the DNA to degrade. However, this may have been because I was heating the solution to room temperature for experimenting each time I came into the lab where my research was conducted. Since I had read that heat causes degradation, I decided to conduct another experiment, called Phase 2.

The results for Phase 2 raised several more questions, too. The percentages of DNA remaining in the solutions were extremely high, even for the control group. The magnetic field, SonikatorÒ , and cooling/heating cycles appeared to cause more degradation in the DNA strands. However, I think this may have been because the DNA was not completely dissolved in the solution before I began my tests. Or, the DNA I used could have already had a high percentage of fragmentation before I put it in the solution. As a result of the questions raised, my hypothesis could be partially accepted and rejected. Therefore, I can conclude my results as inconclusive, at least the experiment is repeated.
 
 

The purpose of this experiment was to determine what different factors cause degradation in Oncorhynchus sp. DNA. There were three variables that I exposed the DNA solution to: extreme coldness, and then to a sudden warmer temperature, a polarity-reversing magnetic field, and a high-frequency Sonikator®. After exposing them to each factor and spooling, I wanted to know the percentage of the concentration of DNA fragments remaining in solution.

When I chose this topic, I was extremely interested in the ways that it could benefit society. Also, this experiment is rare, and original. However, it has evolved a lot in between the time I began researching to the day I started my experiment. In the beginning, I had wanted to work with factors that affected the ability of DNA to spool. Then, I decided to narrow it down to how electromagnetic fields caused the DNA strands to degrade. I had originally planned to use a power supply to create an electromagnetic field. Then, when I wasn't getting much of a field from it, I decided to use an extremely large, circular, permanent magnet. When I reached the lab, however, a unique instrument caught my eye. The instrument was called a magnetic stirrer, which is used to stir substances and other various things. I was watching the stir bar in a beaker containing TE Buffer, and I realized that I could find out if exposing the DNA solution to a situation where the polarity is reversing causes degradation. So, I began my experiment, exposing DNA solutions to the magnetic stirrer for different time intervals. After analyzing the different concentrations for the remaining DNA solutions, I was very surprised by my results. The concentrations of the solutions showed no patterns, or any way to predict the concentration of the upcoming test. Then I thought back to the research I had done, and I remembered that heat causes DNA to degrade. DNA has to be refrigerated to be stored, or it spoils. I had been putting the flask of stock DNA solution in a sink with lukewarm water to get the temperature to rise to that of the room. This action could have caused the DNA to degrade at the same time. I also put a couple samples of DNA solution in a Sonikator®, a device that dissolves solutions by the use of sound waves. This was the turning point of my experiment, making it a stronger project, and teaching me more about scientific methods. So, I decided to order more DNA and complete my experiment in one day without refrigerating the solution.

This experiment will benefit society in several ways. My research could help people experimenting at biotechnological companies to handle DNA safely, without damaging it. DNA is very fragile, and you don't want to destroy it. Placing a DNA solution near common laboratory equipment like a Sonikator®, or a magnetic stirrer may cause it to degrade. Or, taking the DNA solution out of the refrigerator, and allowing it to warm up may cause it to fragment. This is what I'm researching and trying to find an answer for.
 
 

My hypothesis is that heat will most definitely cause an increase in the degradation of the DNA strands of Oncorhynchus sp. DNA. I also believe that allowing the DNA solution to sit on a magnetic stirrer for a given amount of time will cause it to degrade. As for placing samples of the DNA solution in a high-frequency Sonikator®, I think that this may make the DNA strands more susceptible to degradation.

I base my hypothesis on many different sources. One would be an experiment performed by Dr. Henry C. Lai at the University of Washington. In his experiment, he exposed rats to a magnetic field alternating at 60Hz for two hours. He then concluded that there was an increase in the DNA single and double strands that fragmented in their brain cells.

Another source that I base my hypothesis on is my own preliminary research. I had started what I thought would be my experiment for my project, only to find that heat appears to cause the DNA strands to degrade, therefore making it harder to spool. That was my conclusion after repeatedly taking the stock DNA solution out of the refrigerator and warming it to room temperature before testing. My results showed a rapid increase in the concentration of DNA fragments remaining in solution after spooling. This increase occurred with each additional heating/cooling cycle.

Some additional preliminary observations I noted occurred when I put a few samples of the DNA solution in a high-frequency Sonikator® for a time interval of five minutes and another for twenty minutes. Sonikator®s are used in laboratories to dissolve substances in liquids by exposure to sound waves. After spooling and analyzing the remaining solution in the spectrophotometer, there seemed to be evidence that the high-frequency sound waves of the Sonikator® caused degradation in the DNA.

The following constants for both Phases 1 and 2 were:

The DNA used in my experiment was from salmon testes.

2mL DNA solution was used for each test.

  Small, plastic sample bottles, supplied by the Tree Top Technical Center, were used for each test.

A Gilson® Auto-Adjustable Pipette was used to measure the volume of the DNA solution, ethanol, TE buffer, and the DNA solution after spooling.

A different tip was placed on the auto-pipette before using it to measure a different solution.

Five drops of NaCl solution was added to the DNA solution during the spooling process.

5mL of ethanol was added to the DNA and NaCl solution before spooling.

Two wooden sticks were used to spool the DNA out of each solution in the entire experiment, one at a time.

The wooden stick was rotated ten times in the DNA solution before being taken out and disposed of.

After the lid was secured on the DNA solution, the bottle was inverted fifteen times before spooling.

To bring the DNA solution to room temperature, the stock solution was warmed in 22?C water for approximately 10-15 minutes, until reaching room temperature.

The same magnetic stirrer was used to create a rotating magnetic polarity for all trials in both phases.  It was also set at "3" on the dial.

  A spectrophotometer was used to measure the concentration of DNA in the leftover solution for all tests.

The process in using the spectrophotometer to analyze the absorbency was the same for all tests in the experiment.

The process of calculating the concentrations of DNA remaining in the solutions was the same throughout both phases.

All samples within any trial were placed on the magnetic stirrer for the length of time required by their specific trial. (Groups 2-7)

Tests from Groups 2-4 were refrigerated for the same amount of time each for each cycle.

  All samples within any trial were placed in the Sonikator for the length of time required by their specific trial. (Groups 8-10)

Massing 1g of DNA on the analytical balance

Phase 1: Manipulated and Responding Variables

The manipulated variable for Phase 1 was exposing the samples of DNA solution to the oscillating polarity of the magnetic field for several different time intervals. A magnetic stirrer supplied the magnetic field. The solutions did not have a stir bar placed in them, as would any other mixture placed on the stirrer.

The responding variable for Phase 1 was whether or not the magnetic field with the oscillating polarity from the magnetic stirrer caused degradation in Oncorhynchus sp. DNA.

To measure the responding variable, I placed samples of the DNA solution on a magnetic stirrer for six different time intervals. The time intervals were: 30 minutes, 1 hour, 1.5 hours, 3 hours, 27.5 hours, and 36 hours. After exposure, I spooled the DNA strands from the solution samples. Then I used a spectrophotometer to analyze the absorbency of the DNA fragments. After this, I divided the absorbency by the dilution (1.053). Then I multiplied this number by 100 to find the percentage of DNA remaining in the solution.

There are three manipulated variables in this experiment: refrigerating the solution and then gently bringing it to room temperature three different times, placing samples on a magnetic stirrer for three different time intervals, and placing samples in a high-frequency Sonikator® for three different time intervals.

The responding variable was whether or not the DNA strands degraded, or fragmented, after exposing them to the factors listed as the manipulated variables.

To measure the responding variable, I placed samples of the DNA solution on a magnetic stirrer for three different time intervals. The time intervals were 1 hour, 3 hours, and 6 hours. For the test involving the cooling/heating cycles, I separated the stock DNA solution into two different containers and refrigerated one three different times, for one hour each. I then warmed the container in 22° C water. For the tests in the SonikatorÒ , the samples were placed in the device for three different time intervals: 15 minutes, 30 minutes, and 60 minutes. After all tests, I spooled the DNA strands from the solution samples. Then I used a spectrophotometer to analyze the absorbency of the DNA fragments. After this, I divided the absorbency by the dilution (1.090). Then I multiplied this number by 100 to find the percentage of DNA remaining in the solution.
 

Part A: Making the DNA Solution

1. Using an analytical balance, measure 1g of Oncorhynchus DNA.
2. Pour 250 mL TE buffer into a flask.
3. Using forceps, place the DNA into the flask with the TE buffer.
4. Gently swirl the flask around, wetting the DNA.
5. Once the DNA is wet, place a rubber stopper on the flask.
6. Set the flask out at room temperature for at least 48 hours to allow the DNA to dissolve in the mixture.

**STOP**

*If conducting experiments in Phase 1, use only steps from Parts C, & F-J

*If conducting experiments in Phase 2, then follow all steps below with the exception of Part C
 
 

Part B: Refrigeration of DNA Samples

1. After the DNA is completely dissolved in the solution, separate about 1/3 of the solution, and pour it into a separate flask.
2. Place this new solution in the cold store room, at 4ºC.
3. Take the solution out of the refrigerator after one hour.
4. Use the auto-pipette to put 2mL of DNA solution into three different sample bottles.
5. Use the steps for spooling the DNA below.
6. Repeat steps 2-5 two more times for the other two tests.

Part C: Exposure to Polarity-Reversing Magnetic Stirrer, Phase 1

1. Plug in the magnetic stirrer supplied to you, and adjust the speed setting to yield an amplitude of 20 Gauss.
2. To find the amplitude, use a Vernier® Magnetic Sensor. This device, when hooked up to a computer, will measure the amplitude of the magnetic field induced by the magnetic stirrer.
3. Place eighteen sample bottles of the DNA solution on the magnetic stirrer, in two circles, one inside the other. (The amplitude of the field is the same on all parts of the magnetic stirrer)
4. After thirty minutes have passed, take three samples off the stirrer. Label them 'Group 2.'
5. Immediately spool and analyze with spectrophotometer, using the procedures below.
6. After one hour has passed, take three more samples of the DNA solution off the magnetic stirrer.
7. Repeat step five.
8. After an hour and a half take three more samples off the magnetic stirrer.
9. Repeat step five.
10. When three hours have passed, take three more samples off the stirrer. Repeat step five.
11. After twenty-seven and a half hours passed, take three samples off the magnetic stirrer, and repeat step five.
12. Finally, after thirty-six hours, take the last three samples off the magnetic stirrer, and repeat step five.
13. Record all results.

Part D: Exposure to Oscillating-Polarity Magnetic Stirrer, Phase 2

1. Plug in the magnetic stirrer supplied to you, and adjust the speed setting to yield an amplitude of 20 Gauss.
2. To find the amplitude, use a Vernier® Magnetic Sensor. This device, when hooked up to a computer, will measure the amplitude of the magnetic field induced by the magnetic stirrer.
3. Place nine bottles of the DNA solution on the magnetic stirrer, in a circle.
4. After one hour has passed, take three samples of the DNA solution off the magnetic stirrer.
5. Immediately spool the DNA strands from the solution, using the procedures below.
6. Analyze the remaining solutions for the percentage of DNA, using the spectrophotometer.
7. When three hours have passed, take three more samples off the stirrer.
8. Repeat step six.
9. After six hours, take the last three samples off the magnetic stirrer, and immediately spool and analyze using the spectrophotometer.
10. Record your results.

Part E: Exposure to High-Frequency Sound Waves of the Sonikator®

1. Put about 30mL of de-ionized water into the high-frequency Sonikator® and plug it in.
2. Place three sample bottles of DNA solution in the device, in a triangle shape.
3. Turn on the Sonikator®.
4. Allow the solutions to be in the device for one hour.
5. After the hour is up, turn the device off.
6. Take the DNA solutions out and immediately spool.
7. Analyze for fragmentation by using the procedures below for the spectrophotometer.
8. Repeat steps 1-3.
9. Allow the DNA solution samples to be in the Sonikator® for thirty minutes.
10. Turn off the device after thirty minutes has passed.
11. Immediately spool, and then analyze for fragmentation.
12. Again, repeat steps 1-3.
13. Leave the samples in the Sonikator® for only fifteen minutes this time.
14. After fifteen minutes, turn off the Sonikator®.
15. Immediately spool the solutions, and then analyze for the percentage of DNA remaining in solution.

Part F: Spooling the DNA

1. Add five drops of salt solution to the sample bottle containing 2mL of the DNA solution. Use a small, disposable pipette.
2. Using an auto-adjustable pipette, put 5mL of 95% ethanol in the sample bottle. Let it run down the side of the bottle, allowing it to layer on top of the DNA and salt mixture.
3. Secure the lid on the bottle.
4. Very carefully, invert the bottle fifteen times to mix the salt solution, ethanol and the DNA.
5. Using a wooden splint, slowly circle it around the inside of the sample bottle ten times, allowing it to collect all coagulated DNA.
6. Use a second wooden splint to repeat step 5.
7. Repeat steps 1-6 for all trials within each test.

Part G: Preparing Spectrophotometer for Analysis of DNA Degradation

Blank Scan:

*This scan should only be performed once throughout the whole experiment, so long as the experiment is being completed all in one day.

1. Using a pipette, fill a cuvette 2/3 full with the TE buffer.
2. Place it in the spectrophotometer, and be careful not to touch the clear, glass sides of the cuvette. Only touch the cloudy sides, because even a fingerprint can throw off the accuracy of the machine.
3. Secure the cuvette into the spectrophotometer.
4. Press the "scan blank" button on the computer hooked up to the machine.
5. Print a hardcopy of the graph of the blank scan.
6. Take the cuvette out of the spectrophotometer, and pour the buffer into a beaker used for wastes for the time being.

*This is only necessary to perform once throughout the experiment, so long as the spectrophotometer is in constant use by the same solutions.
 
 

1. Use the auto-adjustable pipette that measures up to 5mls to put 2.45mL TE buffer in the cuvette.
2. Use the pipette that measures up to 1mL to add 0.05mL of the stock DNA solution to the cuvette with the buffer.
3. Place a piece of Parafilm® over the cuvette, and invert it several times, allowing the to liquids to mix.
4. Place the cuvette in the spectrophotometer, and secure it.
5. Press the "scan" button on the computer.
6. Print a hardcopy of the graph. The absorbency of the mixture given on the graph will be the number used in the calculations of the concentration of DNA fragments later on in the experiment.
7. Take the cuvette out of the spectrophotometer and pour the mixture into the waste beaker.
8. Put some TE buffer (it doesn't matter what volume) into the cuvette. Place your finger over a sheet of the Parafilm®, and over the cuvette to rinse it out.

Part H: Analysis of DNA Degradation

*These procedures will be used after spooling the DNA strands from the solutions.

1. Adjust a pipette to measure 2.325mls.
2. Put 2.325mL of TE buffer into the cuvette.
3. Adjust a different pipette to measure 0.175mls.
4. Put 0.175mL of DNA solution from one sample bottle into the cuvette.
5. Place a piece of Parafilm® over the cuvette, and invert it several times, allowing the to liquids to mix together.
6. Place the cuvette into the spectrophotometer and secure it in place.
7. At the computer, press the "scan" button. This allows the spectrophotometer to analyze the light absorbed by the DNA fragments, therefore showing the absorbency on a graph.
8. Print a hardcopy of the graph.
9. Back at the computer, press the exit button, and it will take you back to the menu.
10. At the menu, change the wavelength to individual, and then choose to analyze the absorbency at a wavelength from 260 to 0.
11. Press "scan." This will analyze the absorbency of the concentrated DNA at a 260 wavelength. Record the absorbency given to you.
12. Press "scan" twice more.
13. Record your results.
14. Take the cuvette out of the spectrophotometer, and cleanse it by rinsing with TE buffer.
15. Repeat steps 1-14 for all tests.
16. If experimenting is over, cleanse the cuvette with deionized water, dry, and store.

Part I: Calculation of Concentration of DNA Fragmentation

1. To calculate the concentration of DNA fragments, start by averaging the three absorbencies scanned at a 260 wavelength.
2. Divide the average by the dilution of the TE buffer and the stock DNA solution. (The Phase 1 dilution was 1.053, and the Phase 2 was 1.090)
3. Multiply this number by 100 to find the percentage of the DNA remaining in the solution.

Part J: Disposal of DNA

1. Oncorhynchus sp. DNA tissue and solutions will be disposed of after autoclaving, as per Biosafety Level 1 practices described in Biosafety in Microbiological and Biomedical Laboratories, 4^th edition, May 1999.

Results: Phase 1

The original purpose of the experiments in Phase 1 was to determine if exposure to a magnetic field with an oscillating polarity would cause the DNA strands of Oncorhynchus sp. DNA to degrade.

In this experiment, the exposure of the DNA solution to the magnetic field of the magnetic stirrer did cause a dramatic increase in the degradation of the strands. The results below show the average percentage of DNA remaining in the sample solutions after spooling the DNA from all trials within each test.

The percentage of DNA remaining in the solution after spooling strands from the control was 16.9%. After samples were exposed to the oscillating polarity of the magnetic stirrer for thirty minutes, the percentage raised to 44.5%. The samples exposed for one hour and others for an hour and a half both had the same percentage of DNA remaining in solution: 47.4%. The three-hour exposure left the samples with an average of 38.6% of DNA in solution. This was a significantly lower percentage than the others. After being placed on the magnetic stirrer for twenty-seven and a half hours, the average percentage of DNA remaining in the solutions was 47.2%. Finally, the samples that were on the magnetic stirrer for thirty-six hours left 48.1% of DNA remaining in the solution.

Already planning my experiments for Phase 2 of this experiment, I placed three samples into a Sonikator® to find out if the high-frequency sound waves caused the DNA to degrade. The average percentage of DNA remaining in the solution for the three samples was 57.4%.

As an experiment follow-up, I spooled and analyzed the remaining DNA in solution for two more control tests. These samples were both taken from the stock solution that had been refrigerated several more times after testing the original control group. I had a feeling that warming the container of stock solution in warm water until reaching room temperature might have caused it to degrade. The results show that this method of cooling and heating the stock solution does cause the DNA to degrade. The percentage of DNA remaining in the solution for the first follow-up test was 55.0%. As for the second follow-up, the percentage of DNA remaining in the solution was 74.9%.

The original purpose of this experiment was to determine what factors caused the DNA strands of Oncorhynchus sp. DNA to degrade. There were three factors, and the results are printed below. Each percentage is an average from three trials for each test.

To start with, the percentage of DNA remaining in solution after spooling for the control group was 55.8%. This percentage is extremely high, and it means that only about 46% of the DNA strands were spooled out of the solution.

The results of this experiment were varied, and not as expected. After the separate stock DNA solution was placed in the cold store room for the first time, and then slowly brought to room temperature, the percentage of concentrated DNA remaining in solution was 55.5%. This percentage came from Group 2. After the second cycle, the percentage raised to 61.4%. The third and final test of this particular experiment had a percentage of 55.7%.

After spooling the DNA from the samples in Group 5, I found that the percentage of DNA remaining in the solution was 49.3%. This number is extremely low, considering that the percentage for the control group was 55.8%. Group 5 samples were on the magnetic stirrer for one hour. The percentage of DNA remaining in the solution after sitting on the magnetic stirrer for three hours (Group 6) was 60.8%. Finally, tests from Group 7 showed that the percentage of DNA remaining in solution after six hours on the magnetic stirrer was 63.1%.

Group 8 samples were placed in the Sonikator® for fifteen minutes, and after spooling, the percentage of DNA remaining in the solution was 54.2%. This percentage is also less than that of the control group. After thirty minutes in the Sonikator®, samples from Group 9 showed the percentage of DNA remaining in solution to be 55.4%. This is also less than the control's percentage, but only by about 0.4%. In the final trial of this test, known as Group 10, the DNA solution samples spent sixty minutes in the Sonikator®. The percentage of DNA remaining in the solution was 56.4%.
 
 

The last test I conducted in this phase was actually a follow-up from Phase 1. Using the stock DNA solution from Phase 1, which had been stored in the refrigerator, I ran another control test. The test was labeled as Group 11. The results showed a percentage of DNA remaining in the solution of 71.4%. This percentage is significantly higher than the regular control test from Phase 1. That percentage was only 16.9%.

My hypothesis was that the oscillating-polarity magnetic field would cause degradation in the Oncorhynchus DNA.

After analyzing the different percentages for the remaining DNA in each solution, I was very surprised by my results. The concentrations of the solutions showed no patterns, or any way to predict the concentration of the upcoming test. Then I thought back to the research I had done, and I remembered that heat causes DNA to degrade. I also put a couple samples of DNA solution in a Sonikator®, a device that dissolves solutions by the use of sound waves. This was the turning point of my experiment, making it a stronger project, and teaching me more about scientific methods. So, I decided to order more DNA and complete my experiment in one day without refrigerating the solution.

Based on my results, I accept my hypothesis; however, with some caution in doing so. My data shows a dramatic increase in the percentage of DNA remaining in solution after spooling. Many questions are raised by this experiment. During my research, I remember learning that heat can cause DNA strands to fragment. DNA has to be refrigerated to be stored, or it spoils. When I was ready to start experimenting, I repeatedly took the DNA solution from the refrigerator and put the flask in a sink filled with lukewarm water to get the temperature to rise to that of the room. This action could have caused the DNA to degrade at the same time. So, I decided to run two more control tests. I put the DNA stock solution back in the refrigerator for three hours, and then took it out. I warmed it as usual and ran a two more control tests. My results show that heat most likely caused degradation in the DNA strands. This lead me to repeat my experiment, using different factors that can cause DNA to degrade, including the cooling and heating cycles.

My hypothesis was that all three situations the DNA was exposed to would cause degradation. The three situations were exposure to the oscillating-polarity magnetic field for different intervals of time, cooling/heating cycles, and exposure to the high-frequency sound waves.

The results indicate that my hypothesis should be partially accepted and rejected. For Groups 2-4, when DNA samples were refrigerated and then slowly brought to room temperature, the results greatly surprised me. Both the first and third cycles of this method showed a percentage of DNA remaining in solution both less than the control. However, the second cycle showed a percentage of DNA remaining in solution extremely higher than the percentage of DNA remaining in the control group.

As for the tests where the DNA samples were placed in the Sonikator®, I am concluding that this method does cause DNA to degrade. However, all but one test in this particular round turned out to have a percentage of DNA remaining in solution less than the percentage from the control. There was an increase in the percentage of DNA remaining in solution as the time intervals increased.

For the third test in this experiment, I conclude that the oscillating-polarity magnetic field did cause a gradual degradation in the DNA. After spooling, the one-hour exposure showed the percentage of DNA remaining in solution less than the control's. However, the other two trials, the 3-hour and 6-hour exposures, had percentages of DNA remaining in solution much higher than the control's.

Several questions were raised during this experiment. The first thing that came to my mind was that the percentage of DNA left in the control group was way higher than it should have been. I had even repeated the control test several times. I kept coming out with an extremely high percentage of DNA remaining in solution after spooling. Also, I wondered about the accuracy of the second cooling/heating cycle, especially since the percentage of DNA remaining in solution after spooling was so much higher than the percentage from the third cycle. I think that it is definitely a possibility that the DNA was not fully dissolved in the solution before I began my experiment. This could be a big problem, too, because the DNA could have been more concentrated in different parts of the stock solution. Since I separated the DNA stock solution into two different containers for the refrigeration tests, the DNA could have been more concentrated in either one of these. Another possibility could be that the DNA supplied to me could have already had a high percentage of fragmentation before putting it into solution. .

As a result of the questions raised, I plan to repeat this experiment. In repeating the experiment, I will make sure to allow the DNA plenty of time to dissolve in the solution before experimenting. I would also advise others conducting this experiment to have plenty of experience and familiarity with laboratory equipment before beginning.

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