Purpose:
Our purpose was to make 10 milliliters (mL) of 5 NaCl solution, and to make 100mL of TE buffer from 10 mM TRIS, 1 mM EDTA. Also we had to find out if DNA could be spooled out of solution, to find out what DNA looks like and its many unique properties. Finally we needed to find out what yield of DNA can be recovered during the isolation, to prepare and pour an agarose gel for DNA fragment analysis, to find out what the appearance of different DNA samples on an agarose gel.
Materials List:
Lab 4A
Lab 4B
Lab 4I
Lab 4J
Procedure:
Day 1
1. Determine mass of NaCl to be measured to give a 5 M concentration for 10 ml of the solution.
2. Put NaCl in 15 ml conical tube. Add distilled water until solution's volume is 10 ml.
3. Cap 15 ml conical tube. Label and keep in 4 degrees Celsius until ready to use.
4. Determine mass of TRIS to be measured to get correct concentration and volume in TE buffer.
5. Determine mass of EDTA to be measured to get correct concentration and volume in TE buffer.
6. Add TRIS and EDTA in correct amounts to 250-ml.
7. Add 80 ml of deionized water and mix until chemicals dissolved. Add NaOH to raise pH until in range (7.5 and 8.5). Use pH paper to determine pH. If solution too basic, add small volumes of 1 M of HCl to lower pH.
8. Add deionized water until total volume is 100 ml.
9. Pour buffer in 125-ml bottle, cap it, label it, and store ate 4 degrees Celsius until ready to use.
Day 2
1. Mix 1 ml of DNA with 1 ml of TE buffer.
2. Dilute DNA with TE in beaker; observe.
3. Add NaCl (500 microliters of 5M NaCl).
4. Add 4 ml of Ethanol (trickle down side so careful not to mix solutions); observe.
5. Spool DNA.
6. Put DNA into new tube with 2 ml fresh TE and label.
Day 3
1. Determine the mass of agarose to be measured to put in the 1X TAE (TRIS-acetate -EDTA).
2. Add agarose to 100 ml 1X TAE in erlenmeyer flask.
3. Heat to boil and dissolve (heat-swirl-heat-swirl until clear) in a microwave oven.
4. Let cool until you can touch the erlenmeyer flask for a few seconds.
5. Prepare the gel mold by taping the opposite open ends and putting the two combs in.
6. Pour 1X TAE and agarose solution into gel mold and let cool.
Day 4
1. Remove tape from gel. Place in gel tank.
2. Pour TAE over gel until covered. Gently remove combs.
3. Prepare samples with micropipet (P-2-20):
- 20 ml DNA and 4 microliters 6x loading dye. Spin 2 seconds in centrifuge.
4. Load samples onto gel with micropipet (P-20-200).
5. Put cover on gel tank and plug into power supply.
6. Run at 110V for approximately 45 minutes.
7. Stain for several hours with Ethidium Bromide
8. Rinse and observe with light.
Data Analysis
Sadly, our experiment failed and we ended with no results other than it failed. There are a variety of reasons that it could of failed.It was stained overnight and the DNA could have diffused out. The DNA denatured, dye not loaded correctly,dye did not resuspend before loading in gel, buffers not made correctly, one of the reagents was bad. All these reasons could have contributed to the results of our experiment failing.We then analyzed each of these reasons and came up with a probability of them being the factor. The DNA diffusing out is not likely because it is too big. The stains purpose is to denature the DNA so it it is also unlikely to be the cause. IF the die was not loaded correctly, or the dye was not resuspended before loading in the gel, or the buffers were made incorrectly, are all not likely to happen to the whole class . So finally that left us with one of the reagents were bad. This is very possible since it is life sensitive and it could have broken down from the light exposure,
Our teacher Dr.LB remade the stock solution, and was able to find that the stock solution was the reason of the failed experiment. She was able to come to this conclusion by remaking the stock solution one with the new and one with the old, and she saw the older solution failed. We then tested it on our DNA gel and were able to see where our DNA was and save our experiment.
Our purpose was to make 10 milliliters (mL) of 5 NaCl solution, and to make 100mL of TE buffer from 10 mM TRIS, 1 mM EDTA. Also we had to find out if DNA could be spooled out of solution, to find out what DNA looks like and its many unique properties. Finally we needed to find out what yield of DNA can be recovered during the isolation, to prepare and pour an agarose gel for DNA fragment analysis, to find out what the appearance of different DNA samples on an agarose gel.
Materials List:
Lab 4A
- analytical balance
- tabletop milligram balance
- weigh paper
- weigh boat
- lab scoops
- sodium chloride
- 15 ml tubes
- tube racks for 15 ml tubes
- TRIS
- EDTA
- 125 ml bottle
- 100 ml graduated cylinder
- pH paper, wide/narrow-range
- Hydrochloric acid
- Sodium hydroxide
- glass rods
Lab 4B
- 50 ml beakers
- salmon sperm DNA
- 2 - 20 ml pipet
- pipet pump
- micropipet (P-1000)
- micropipet tips for P-1000
- ethanol (95%)
- glass rods
- 15 ml conical tubes (capped)
- tube racks fro 15 ml conical tubes
- permanent lab markers
Lab 4I
- TAE buffer concentrate (40x stock)
- 600 ml beaker
- agarose
- tabletop milligram balance
- weigh paper
- lab scoops
- 250 ml media bottle
- permanent lab markers
- microwave oven
- hot hands protector
- horizontal gel box for agarose gels
- 50 ml beakers
Lab 4J
- horizontal Gel box for agarose gels
- prepared agarose gel
- TAE buffer concentrate (40x stock)
- tube rack for 15 ml conical tubes
- permanent lab markers
- DNA samples
- gel loading dye (6x)
- micropipet (P-2-20)
- micropipet (P-20-200)
- micropipet tips for P-2-20
- micropipet tips for P-20-200
- microcentrifuge
- power supply
- ethidium bromide (0.5 micrograms/ml)
- gloves
Procedure:
Day 1
1. Determine mass of NaCl to be measured to give a 5 M concentration for 10 ml of the solution.
2. Put NaCl in 15 ml conical tube. Add distilled water until solution's volume is 10 ml.
3. Cap 15 ml conical tube. Label and keep in 4 degrees Celsius until ready to use.
4. Determine mass of TRIS to be measured to get correct concentration and volume in TE buffer.
5. Determine mass of EDTA to be measured to get correct concentration and volume in TE buffer.
6. Add TRIS and EDTA in correct amounts to 250-ml.
7. Add 80 ml of deionized water and mix until chemicals dissolved. Add NaOH to raise pH until in range (7.5 and 8.5). Use pH paper to determine pH. If solution too basic, add small volumes of 1 M of HCl to lower pH.
8. Add deionized water until total volume is 100 ml.
9. Pour buffer in 125-ml bottle, cap it, label it, and store ate 4 degrees Celsius until ready to use.
Day 2
1. Mix 1 ml of DNA with 1 ml of TE buffer.
2. Dilute DNA with TE in beaker; observe.
3. Add NaCl (500 microliters of 5M NaCl).
4. Add 4 ml of Ethanol (trickle down side so careful not to mix solutions); observe.
5. Spool DNA.
6. Put DNA into new tube with 2 ml fresh TE and label.
Day 3
1. Determine the mass of agarose to be measured to put in the 1X TAE (TRIS-acetate -EDTA).
2. Add agarose to 100 ml 1X TAE in erlenmeyer flask.
3. Heat to boil and dissolve (heat-swirl-heat-swirl until clear) in a microwave oven.
4. Let cool until you can touch the erlenmeyer flask for a few seconds.
5. Prepare the gel mold by taping the opposite open ends and putting the two combs in.
6. Pour 1X TAE and agarose solution into gel mold and let cool.
Day 4
1. Remove tape from gel. Place in gel tank.
2. Pour TAE over gel until covered. Gently remove combs.
3. Prepare samples with micropipet (P-2-20):
- 20 ml DNA and 4 microliters 6x loading dye. Spin 2 seconds in centrifuge.
4. Load samples onto gel with micropipet (P-20-200).
5. Put cover on gel tank and plug into power supply.
6. Run at 110V for approximately 45 minutes.
7. Stain for several hours with Ethidium Bromide
8. Rinse and observe with light.
Data Analysis
Sadly, our experiment failed and we ended with no results other than it failed. There are a variety of reasons that it could of failed.It was stained overnight and the DNA could have diffused out. The DNA denatured, dye not loaded correctly,dye did not resuspend before loading in gel, buffers not made correctly, one of the reagents was bad. All these reasons could have contributed to the results of our experiment failing.We then analyzed each of these reasons and came up with a probability of them being the factor. The DNA diffusing out is not likely because it is too big. The stains purpose is to denature the DNA so it it is also unlikely to be the cause. IF the die was not loaded correctly, or the dye was not resuspended before loading in the gel, or the buffers were made incorrectly, are all not likely to happen to the whole class . So finally that left us with one of the reagents were bad. This is very possible since it is life sensitive and it could have broken down from the light exposure,
Our teacher Dr.LB remade the stock solution, and was able to find that the stock solution was the reason of the failed experiment. She was able to come to this conclusion by remaking the stock solution one with the new and one with the old, and she saw the older solution failed. We then tested it on our DNA gel and were able to see where our DNA was and save our experiment.
Conclusion:
Isolating DNA can be called purifying the DNA, it is a process that is very complicated. In order to receive an accurate results through DNA testing, the DNA can not be in anyway contaminated. Scientists can analyze DNA when it is purified by using the gel as a medium for the DNA. A gel can suppress thermal convection and delay the passage of molecules. All in all, it maintains separation so it can be stained with a reagent after the isolation. Isolating DNA and separting it in a gel is quite frequently used in several fields of science. It can be used by making a sample of DNA to tell if something contains a disease, and or cancer.
Reflection:
During the course of this project me and my partners did amazing together. Rosalie, Dalton, Tony, Zach, Ingrid, Cole, and I were able to work efficiently together and able to get along very well. Although we could have worked more efficiently by helping each other with the problems instead of just doing it our selves, we still got the job done. In the future by slowing things down and working harder we can do a better job at learning all these new skills. For instance,the majority of my group and I were really great at pipeting, I believe there was only one of use who couldn't pipet very well. Instead of us doing it for that person, we should of taught them how to do it so in the future they can be successful in pipeting. My group was very successful in making all the solutions and adjusting the pH level to what it needed to be. I believe that we do not need to practice more, but it wouldn't hurt. All in all, I loved this lab, I have to say it was the most interesting and entertaining lab we have done all semester.
Isolating DNA can be called purifying the DNA, it is a process that is very complicated. In order to receive an accurate results through DNA testing, the DNA can not be in anyway contaminated. Scientists can analyze DNA when it is purified by using the gel as a medium for the DNA. A gel can suppress thermal convection and delay the passage of molecules. All in all, it maintains separation so it can be stained with a reagent after the isolation. Isolating DNA and separting it in a gel is quite frequently used in several fields of science. It can be used by making a sample of DNA to tell if something contains a disease, and or cancer.
Reflection:
During the course of this project me and my partners did amazing together. Rosalie, Dalton, Tony, Zach, Ingrid, Cole, and I were able to work efficiently together and able to get along very well. Although we could have worked more efficiently by helping each other with the problems instead of just doing it our selves, we still got the job done. In the future by slowing things down and working harder we can do a better job at learning all these new skills. For instance,the majority of my group and I were really great at pipeting, I believe there was only one of use who couldn't pipet very well. Instead of us doing it for that person, we should of taught them how to do it so in the future they can be successful in pipeting. My group was very successful in making all the solutions and adjusting the pH level to what it needed to be. I believe that we do not need to practice more, but it wouldn't hurt. All in all, I loved this lab, I have to say it was the most interesting and entertaining lab we have done all semester.