Lab 4: Exploring Deoxyribonucleic Acids (DNA)
Materials:
- Analytical Balance - Tabletop Milligram Balance - 7.6 x 7.6 cm Weigh Paper - 3.5 x 3.5 Weigh Boat - Lab Scoops - Sodium Chloride -15 mL Capped Tubes - Tube Racks - TRIS - EDTA - Disodium Salt - 125 mL Bottle - 100 mL Graduated Cylinder - Glasses |
- pH Paper - Hydrochloric Acid - Sodium Hydroxide - Glass Rods - 50 mL Beakers - Salmon Sperm DNA - 2 mL Pipette - P-1000 Micropipette, and tips - 95% Ethanol - Permanent Marker Pens - 1L Tripour Plastic Peaker - 40X TAE Buffer Concentrate - 600 mL Beakers - Agarose |
- 250 mL Media Bottle - Microwave Oven - Hot Hands Protector - Horizontal Gel Box - 65 degree Celcius water bath - Reaction Tubes, 1.7 mL - DNA Samples - Loading Dye: 6x concentration - Micro Pipettes - Microcentrifuge - Power Supply - Ethidium Bromide - Gel Photo Imaging System - Gloves |
Lab 4a & b: Creating DNA Solutions & Spooling DNA
Purpose: In the first part of this lab, we created two different solutions by adding several substances within certain parameters and proportions. We made a 10 mL solution of 5 mole NaCl; a 100 mL solution of 10 micro mole Tris; and a 1 micro mole solution of EDTA.
Hypothesis: We hypothesized that if you added the correct amount of solution, you would be able to spool a substantial amount of DNA.
Hypothesis: We hypothesized that if you added the correct amount of solution, you would be able to spool a substantial amount of DNA.
Conversions to Liters and Moles:
10 mL= 0.01 L
10 mM= 0.01 M; 100 mL= 0.1 L
1 mM= 0.001 M; 100 mL= 0.1 L
10 mL= 0.01 L
10 mM= 0.01 M; 100 mL= 0.1 L
1 mM= 0.001 M; 100 mL= 0.1 L
Procedure:
1. Make molarity calculations.
[Given: 1 mole = 6.02 x 10^23 ; 5 moles = 5 (6.02 x 10^23 particles) / 1 Litre ; (Molarity) (Volume) (Formula weight) = g substance needed]
Solution One: NaCl: 5M x 0.010 L x 58.44 g/mole = 2.92 grams
Solution Two: TRIS: 0.01 M x 0.1 L x 372.24 g/mole = 0.158 grams
Solution Three: 0.001 M x 0.1 L x 372.24 g/mole = 0.037 grams
2. Create TE Solution by combining Solutions Two and Three
3. Dilute DNA with TE solution in a flask.
4. Add 2.92 grams of NaCl.
5. Add 4 mL of alcohol by trickling it down the side of the flask.
6. Spool DNA
7. Put the spooled DNA into a new tube and add 2 mL of fresh TE solution.
1. Make molarity calculations.
[Given: 1 mole = 6.02 x 10^23 ; 5 moles = 5 (6.02 x 10^23 particles) / 1 Litre ; (Molarity) (Volume) (Formula weight) = g substance needed]
Solution One: NaCl: 5M x 0.010 L x 58.44 g/mole = 2.92 grams
Solution Two: TRIS: 0.01 M x 0.1 L x 372.24 g/mole = 0.158 grams
Solution Three: 0.001 M x 0.1 L x 372.24 g/mole = 0.037 grams
2. Create TE Solution by combining Solutions Two and Three
3. Dilute DNA with TE solution in a flask.
4. Add 2.92 grams of NaCl.
5. Add 4 mL of alcohol by trickling it down the side of the flask.
6. Spool DNA
7. Put the spooled DNA into a new tube and add 2 mL of fresh TE solution.
What is precipitation?
- definition: "taking something out of the solution"
- the precipitate is what doesn't dissolve in a solution
What are DNases?
- enzymes that break down DNA
Why use NaCl?
- sodium ions are positively charged (Na+)
- sodium interacts with the phosphate backbone and makes DNA molecules come closer and thereby clump together, which helps it form a precipitate
Why use TRIS?
- maintains the pH of the solution (7 - 8)
- if the solution is too basic or too acidic, the macro-molecules will "denature," or come apart, defeating the purpose of the previous precipitation
Why use EDTA?
- EDTA is a preservative
- prevents DNase activity
- binds to calcium and magnesium ions, which are co-factors of enzymes that break down proteins and DNA
- for example, food products have EDTA to keep from degrading, rotting
- definition: "taking something out of the solution"
- the precipitate is what doesn't dissolve in a solution
What are DNases?
- enzymes that break down DNA
Why use NaCl?
- sodium ions are positively charged (Na+)
- sodium interacts with the phosphate backbone and makes DNA molecules come closer and thereby clump together, which helps it form a precipitate
Why use TRIS?
- maintains the pH of the solution (7 - 8)
- if the solution is too basic or too acidic, the macro-molecules will "denature," or come apart, defeating the purpose of the previous precipitation
Why use EDTA?
- EDTA is a preservative
- prevents DNase activity
- binds to calcium and magnesium ions, which are co-factors of enzymes that break down proteins and DNA
- for example, food products have EDTA to keep from degrading, rotting
Lab 4i: Making Agarose Gels for Separating and Analyzing DNA Fragments
Calculations: (C1)(V1) = (C2)(V2)
1x TAE Buffer- (40x)(V) = (500 mL)(1x) V1 =12.5 mL
Agaorse- (0.008)(50 mL) = 0.4 grams
1x TAE Buffer- (40x)(V) = (500 mL)(1x) V1 =12.5 mL
Agaorse- (0.008)(50 mL) = 0.4 grams
Procedure:
1. Prepare 100 mL solution of 0.8% agarose in 1X TAE buffer solution.
2. Weigh out the required mass of powdered agarose in a weigh boat. Add it to a 250-mL media bottle.
3. Measure out enough TAE buffer to prepare a total of 100 mL of agarose and buffer mixed together. Swirl to mix.
4. Cap the media bottle and swirl the flask to suspend the agarose in the buffer.
5. To dissolve the agarose, microwave for 4 minutes at 50% power. Wait for the solution to boil.
6. Place the hot dissolved agarose solution on a fireproof lab tabletop and let cool to 65 degrees Celsius before pouring into a gel tray.
7. Place a six-well comb into the notches at the end of the gel tray. This will create the necessary wells for the next experiment.
8. Place the gel (on the gel tray) into a gel box.
9. Pour 1X TAE buffer into the gel box and completely submerge the gel.
10. Gently pull the comb out of the gel and make sure the wells are not broken or cracked.
1. Prepare 100 mL solution of 0.8% agarose in 1X TAE buffer solution.
2. Weigh out the required mass of powdered agarose in a weigh boat. Add it to a 250-mL media bottle.
3. Measure out enough TAE buffer to prepare a total of 100 mL of agarose and buffer mixed together. Swirl to mix.
4. Cap the media bottle and swirl the flask to suspend the agarose in the buffer.
5. To dissolve the agarose, microwave for 4 minutes at 50% power. Wait for the solution to boil.
6. Place the hot dissolved agarose solution on a fireproof lab tabletop and let cool to 65 degrees Celsius before pouring into a gel tray.
7. Place a six-well comb into the notches at the end of the gel tray. This will create the necessary wells for the next experiment.
8. Place the gel (on the gel tray) into a gel box.
9. Pour 1X TAE buffer into the gel box and completely submerge the gel.
10. Gently pull the comb out of the gel and make sure the wells are not broken or cracked.
Lab 4j: Using Gel Electrophoresis to Study DNA Molecules
Procedure:
1. Prepare the gel and gel box for loading.
2. Carefully secure gates of the gel tray. Make sure the 1X TAe buffer covers the gel by at least 1 centimeter.
3. Obtain 1.7-mL tubes.
4. Add 20 micro liters of salmon sperm DNA and add 3 micro liters of 6X DNA loading dye. Spin the sample in a minicentrifuge.
5. Load the salmon sperm DNA sample into the wells using a micro pipette.
6. Connect the electrodes of the gel box to the power supply and run the gel at 110 Volts for 45 minutes.
7. Run until you can see the front loading dye halfway down the gel.
8. Cover the gel with EtBr (ethinium bromide) and stain the gel for 20 minutes. Then pour off the EtBr, cover with deionized water, and observe the gel on a UV light box.
9. Analyze the contents of each well, size of the standards, the samples, and the DNA bands.
1. Prepare the gel and gel box for loading.
2. Carefully secure gates of the gel tray. Make sure the 1X TAe buffer covers the gel by at least 1 centimeter.
3. Obtain 1.7-mL tubes.
4. Add 20 micro liters of salmon sperm DNA and add 3 micro liters of 6X DNA loading dye. Spin the sample in a minicentrifuge.
5. Load the salmon sperm DNA sample into the wells using a micro pipette.
6. Connect the electrodes of the gel box to the power supply and run the gel at 110 Volts for 45 minutes.
7. Run until you can see the front loading dye halfway down the gel.
8. Cover the gel with EtBr (ethinium bromide) and stain the gel for 20 minutes. Then pour off the EtBr, cover with deionized water, and observe the gel on a UV light box.
9. Analyze the contents of each well, size of the standards, the samples, and the DNA bands.
Conclusion/Analysis:
Although our results were non-conclusive, we learned a lot about both DNA isolation and running gels. We were not able to see any DNA activity, which could have occurred because of several reasons:
1. The staining time could have been too short.
- This is unlikely, because it should have worked within the time frame that we stained the gel.
2. We did not re-suspend the solution, therefore failing to acquire sufficient DNA in our sample.
3. We did not make our solutions correctly.
- This is extremely unlikely because all the gels did not run the DNA, and the chances of every person making their solution incorrectly is slim to none.
4. The Ethinium Bromide had spoiled over time.
- This is possible because the EtBr was a year old, and had been recycled from last year's experiment.
5. The DNA diffused off of the gels.
- This is highly unlikely because the DNA macromolecules were too large to travel that quickly through the gel.
6. One of the reagents went bad.
- This is possible because the stain is light sensitive, and could have broken down.
Although our results were non-conclusive, we learned a lot about both DNA isolation and running gels. We were not able to see any DNA activity, which could have occurred because of several reasons:
1. The staining time could have been too short.
- This is unlikely, because it should have worked within the time frame that we stained the gel.
2. We did not re-suspend the solution, therefore failing to acquire sufficient DNA in our sample.
3. We did not make our solutions correctly.
- This is extremely unlikely because all the gels did not run the DNA, and the chances of every person making their solution incorrectly is slim to none.
4. The Ethinium Bromide had spoiled over time.
- This is possible because the EtBr was a year old, and had been recycled from last year's experiment.
5. The DNA diffused off of the gels.
- This is highly unlikely because the DNA macromolecules were too large to travel that quickly through the gel.
6. One of the reagents went bad.
- This is possible because the stain is light sensitive, and could have broken down.
How can we test if the Ethinium Bromide was spoiled?
1. Make a new batch of stain: 20,000x stock - diluted to 1x
a) Remake 1x solution from old 20,000x stock
b) remake 20,000x stock; dilute to make new 1x
2. Stain one gel with each new solution for three hours.
3. Check progress.
1. Make a new batch of stain: 20,000x stock - diluted to 1x
a) Remake 1x solution from old 20,000x stock
b) remake 20,000x stock; dilute to make new 1x
2. Stain one gel with each new solution for three hours.
3. Check progress.
After re-staining the gels, we were able to get our results (as shown in the first picture on this page). We could clearly see that the DNA molecules had moved in their lanes.
Reflection:
DNA is an extremely important macromolecule that scientists have exhaustively researched since its discovery in hopes to better understand life itself. Almost every single biotechnological and biological field deals with DNA in some respect. Unraveling the mysteries of the double-helix has led to medical advancements, and the creation of antibiotics, medicines, and cures. The ability to isolate DNA for study, without the hindrance of other cell organelles, is crucial in order for scientists to receive accurate results. Running gels, and being able to measure out exact solutions, is also paramount for a scientist. In the lab, most experiments involve these skills, so it was a great eye-opener to experience what a day in a real workplace would be like.
Our group collaborated very well, and we always remained on task. We all did our own work, so we each benefited from the learning experience, while at the same time comparing and discussing our thoughts. I would have liked us to manage our time better: we tended to flock through each step and station as a complete group, when we could have split the group and finished two steps at once.
DNA is an extremely important macromolecule that scientists have exhaustively researched since its discovery in hopes to better understand life itself. Almost every single biotechnological and biological field deals with DNA in some respect. Unraveling the mysteries of the double-helix has led to medical advancements, and the creation of antibiotics, medicines, and cures. The ability to isolate DNA for study, without the hindrance of other cell organelles, is crucial in order for scientists to receive accurate results. Running gels, and being able to measure out exact solutions, is also paramount for a scientist. In the lab, most experiments involve these skills, so it was a great eye-opener to experience what a day in a real workplace would be like.
Our group collaborated very well, and we always remained on task. We all did our own work, so we each benefited from the learning experience, while at the same time comparing and discussing our thoughts. I would have liked us to manage our time better: we tended to flock through each step and station as a complete group, when we could have split the group and finished two steps at once.