DNA Labs
Purpose:
To make 10 milliliters (mL) of 5 NaCl solution, to make 100mL of TE buffer: 10 mM TRIS, 1 mM EDTA (DNA storage solution), to find out if DNA an be spooled out of solution, to find out what DNA looks like, to find out its many unique properties, 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:
Procedure:
part I: preperation of 5 M of NaCl
1. determine the mass of NaCl to be measured. In notebook draw and explain a diagram of the solution.
2. place NaCl in a 15mL capped, conical tube. Cap it. Slowly add H2O, while stirring, until a final volume of the 10mL is reached.
3. pour the mixture into a 15mL capped, conical tube. Cap it. Label it with the sample name, concentration, date, and technicians initials. Store at 4 celcius until ready to use.
part II: preperation of TE buffer
1. determine the mass of TRIS to be measured to give the correct concentration and volume in the final TE buffer. Show final calculations in your notebook.
2. determine the mass of EDTA to be measured to give correct concentration and volume in the final TE buffer. Record in notebook.
3. measure out the TRIS and EDTA, and add them to a 250mL beaker.
dna spooling
1. using the TE buffer as the solvent and the C1 V1 = C2 V2 equation, determine how to make 2mL of 2mg/mL from a 4mg/mL salmon sperm DNA solution. Record in notebook.
2. prepare the diluted salmon sperm DNA solution in a prechilled, clean 50mL beaker. You will be using this 2mg/mL DNA solution in the next step.
3. describe the appearance, color, vicoscity, etc, of the 2mg/mL slamon sperm DNA. Add these data to the data table.
4. using a micro pipet, add 500uL of 5 M NaCl solution. Mix by swirling.
5. keep everything as cold as possible. Slowly trickle 4mL of ETOH down the side of the beaker containing the DNA and NaCl. Do not mix the alcohol and DNA layers.
6. observe the interface between the two solutions. You should see a layer of alcohol on the top of the layer containing the DNA and NaCl. Do not mix the two layers. Describe what the layers look like. Add these data to the data table.
7. place the glass rod at the interface of the two layered solution. During spooling, you will force ETOH from the top layer down into the bottom DNA layer and pull the DNA out as it mobes away from the ETOH.
8. holding the beaker tilted toe the right 45degrees, wind the DNA that comes out of the solution onto the rod. these are not single DNA molecules, but thousands of molecules. watch the interface as you rotate the rod.
9. examine and touch the DNA on the rod. Record the appearance of DNA, including color, texture, and other characteristics. Add these data to the data table. Touching the samples exposes it to DNase enzyme, which chops DNA. Record.
10. shake the ethanol of the spooled DNA. Touch it to a paper towel. Get rid of as much ethanol as you can without losing the DNA sample.
11. scrape the spooled DNA into 2mL of TE buffer in a labeled, capped, conical, 15mL tube. Record
12. store at 4degrees c for at least one week. During that time, the DNA should go back into the solution. Record. After that it should be ready to use for indicator testing or gel electrophoresis.
procedure-
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.
procedure-
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.
To make 10 milliliters (mL) of 5 NaCl solution, to make 100mL of TE buffer: 10 mM TRIS, 1 mM EDTA (DNA storage solution), to find out if DNA an be spooled out of solution, to find out what DNA looks like, to find out its many unique properties, 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:
- analytical balance
- tabletop milligram balance
- weigh paper
- weigh boat
- lab scoops
- sodium chloride
- 15 ml conical tubes
- tube racks for 15 ml conical tubes
- TRIS
- EDTA (disodium salt)
- 125 ml bottle
- 100 ml graduated cylinder
- pH paper, wide/narrow-range
- Hydrochloric acid
- Sodium hydroxide
- glass rods
- 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
- 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
- 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:
part I: preperation of 5 M of NaCl
1. determine the mass of NaCl to be measured. In notebook draw and explain a diagram of the solution.
2. place NaCl in a 15mL capped, conical tube. Cap it. Slowly add H2O, while stirring, until a final volume of the 10mL is reached.
3. pour the mixture into a 15mL capped, conical tube. Cap it. Label it with the sample name, concentration, date, and technicians initials. Store at 4 celcius until ready to use.
part II: preperation of TE buffer
1. determine the mass of TRIS to be measured to give the correct concentration and volume in the final TE buffer. Show final calculations in your notebook.
2. determine the mass of EDTA to be measured to give correct concentration and volume in the final TE buffer. Record in notebook.
3. measure out the TRIS and EDTA, and add them to a 250mL beaker.
dna spooling
1. using the TE buffer as the solvent and the C1 V1 = C2 V2 equation, determine how to make 2mL of 2mg/mL from a 4mg/mL salmon sperm DNA solution. Record in notebook.
2. prepare the diluted salmon sperm DNA solution in a prechilled, clean 50mL beaker. You will be using this 2mg/mL DNA solution in the next step.
3. describe the appearance, color, vicoscity, etc, of the 2mg/mL slamon sperm DNA. Add these data to the data table.
4. using a micro pipet, add 500uL of 5 M NaCl solution. Mix by swirling.
5. keep everything as cold as possible. Slowly trickle 4mL of ETOH down the side of the beaker containing the DNA and NaCl. Do not mix the alcohol and DNA layers.
6. observe the interface between the two solutions. You should see a layer of alcohol on the top of the layer containing the DNA and NaCl. Do not mix the two layers. Describe what the layers look like. Add these data to the data table.
7. place the glass rod at the interface of the two layered solution. During spooling, you will force ETOH from the top layer down into the bottom DNA layer and pull the DNA out as it mobes away from the ETOH.
8. holding the beaker tilted toe the right 45degrees, wind the DNA that comes out of the solution onto the rod. these are not single DNA molecules, but thousands of molecules. watch the interface as you rotate the rod.
9. examine and touch the DNA on the rod. Record the appearance of DNA, including color, texture, and other characteristics. Add these data to the data table. Touching the samples exposes it to DNase enzyme, which chops DNA. Record.
10. shake the ethanol of the spooled DNA. Touch it to a paper towel. Get rid of as much ethanol as you can without losing the DNA sample.
11. scrape the spooled DNA into 2mL of TE buffer in a labeled, capped, conical, 15mL tube. Record
12. store at 4degrees c for at least one week. During that time, the DNA should go back into the solution. Record. After that it should be ready to use for indicator testing or gel electrophoresis.
procedure-
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.
procedure-
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.
This is our second try.
Data Analysis
Unfortunately, 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 or other reasons.
Data Analysis part 2
Now that we updated our materials that we thought affected the experiment and compromised it, we retried the process and were successful. The second gel that we made demonstrated the desired results.
Conclusion
This lab we conducted is very useful in our future lives if we chose science. Isolating DNA, or also known as purifying the DNA, is a very valuable process. To receive accurate results in any kind of DNA test, the DNA cannot be contaminated. Scientists can analyze DNA when it is purified. We used the gel as a medium for the DNA. First of all, a gel can suppress thermal convection. Secondly, it can delay the passage of molecules. Finally, it maintains separation so it can be stained with a reagent (ethidium bromide) after the electrophoresis. Isolating DNA and separting it in a gel is quite frequently used in several fields of science.
Reflection
During the course of this project me and my partners did amazing together. Rosalie, Dalton, Tony, Gabby, 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. I really enjoyed these DNA related labs and hope to do some more later in the year.
Unfortunately, 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 or other reasons.
Data Analysis part 2
Now that we updated our materials that we thought affected the experiment and compromised it, we retried the process and were successful. The second gel that we made demonstrated the desired results.
Conclusion
This lab we conducted is very useful in our future lives if we chose science. Isolating DNA, or also known as purifying the DNA, is a very valuable process. To receive accurate results in any kind of DNA test, the DNA cannot be contaminated. Scientists can analyze DNA when it is purified. We used the gel as a medium for the DNA. First of all, a gel can suppress thermal convection. Secondly, it can delay the passage of molecules. Finally, it maintains separation so it can be stained with a reagent (ethidium bromide) after the electrophoresis. Isolating DNA and separting it in a gel is quite frequently used in several fields of science.
Reflection
During the course of this project me and my partners did amazing together. Rosalie, Dalton, Tony, Gabby, 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. I really enjoyed these DNA related labs and hope to do some more later in the year.