DNA Lab
Purpose
Make 100 ml of MNaCl solution.
Make 100 ml of TE buffer.
Find out if DNA can be spooled, what it looks like, what amount of DNA can be recovered, and what are its unique properties.
Prepare and pour an agarose gel for DNA fragment analysis.
Find the appearance of different DNA samples on agarose gel.
Materials
Make 100 ml of MNaCl solution.
Make 100 ml of TE buffer.
Find out if DNA can be spooled, what it looks like, what amount of DNA can be recovered, and what are its unique properties.
Prepare and pour an agarose gel for DNA fragment analysis.
Find the appearance of different DNA samples on agarose gel.
Materials
Analytical Balance
Tabletop Balance Weigh Paper/Boat Scoops Sodium Chloride Capped Tubes(15ml) and Rack TRIS EDTA |
Sodium Hydrochloric Acid
Sodium Hydroxide Granulated Cylinder(100ml) pH strips 50ml Beakers DNA(Salmon Testes) 2ml Pipet and Pump Micropipet, P-1000, and Tips 95% Ethanol |
Glass Rods
TAE buffer, 40X 600ml Beakers Agarose 250ml bottle Microwave Oven Hot Hands Protector Safety Glasses |
Gel Box
Gel Tank Power Supply Gloves Ethidium Bromide Microcentrifuge 6x Loading Dye Distilled Water |
Procedure
Lab 4a
Part 1
1. Measure out 2.92 g of NaCl and add to tube
2. QS water to (add water until you reach) 10 mL
3. Label for later use
Part 2
1. Add 0.1576 g of TRIS and 0.037224 g of EDTA to a beaker
2. Add 80 mL of deionized water to solution
3. Add HCl to raise pH or NaOH to lower pH until solution has a pH between 7.5 and 8.5
4. QS water to 100 mL
5. Label for later use
Lab 4b
1. Add 1 mL DNA and 1 mL TE into a beaker.
2. Add 5M NaCl
3. Add 4 mL ETOH by pouring down side
4. Using a glass rod, spool DNA
5. Place DNA in a tube with 2 mL TE
Lab 4i
1. Add 0.4g agarose powder and QS TAE to 100 mL
2. Heat solution until agarose is dissolved.
3. Let solution cool and then pour into gel box
Lab 4j
1. Submerge gel and electrodes in TAE
2. Add 20 uL DNA and 4 mL of 6x loading dye into a tube
3. Centrifuge solution
4. Load solution into gel
5. Apply charge to gel for approximately 45 minutes
6. Stain with Ethidium Bromide, rinse, and observe
Lab 4a
Part 1
1. Measure out 2.92 g of NaCl and add to tube
2. QS water to (add water until you reach) 10 mL
3. Label for later use
Part 2
1. Add 0.1576 g of TRIS and 0.037224 g of EDTA to a beaker
2. Add 80 mL of deionized water to solution
3. Add HCl to raise pH or NaOH to lower pH until solution has a pH between 7.5 and 8.5
4. QS water to 100 mL
5. Label for later use
Lab 4b
1. Add 1 mL DNA and 1 mL TE into a beaker.
2. Add 5M NaCl
3. Add 4 mL ETOH by pouring down side
4. Using a glass rod, spool DNA
5. Place DNA in a tube with 2 mL TE
Lab 4i
1. Add 0.4g agarose powder and QS TAE to 100 mL
2. Heat solution until agarose is dissolved.
3. Let solution cool and then pour into gel box
Lab 4j
1. Submerge gel and electrodes in TAE
2. Add 20 uL DNA and 4 mL of 6x loading dye into a tube
3. Centrifuge solution
4. Load solution into gel
5. Apply charge to gel for approximately 45 minutes
6. Stain with Ethidium Bromide, rinse, and observe
Data Analysis
Our experiment failed pretty bad. The stain we used did not work in showing the DNA in the gel. We brainstormed about why it did not work and the most likely solution is that one of the reactants in the dye failed to work. We are going to test this theory by making a new batch of dye from a new stock, and we will hopefully be able to see the results we wanted to. The dye worked and we were able to see that our DNA strands are large due to the DNA being higher up in the gel. Conclusion There is immense value in being able to separate DNA on a gel. This technique could be used to help solve heinous crimes of the worst type, and it can provide precious research materials for disease or hereditary research. |
Reflection
My group worked decently well. We could have been more efficient by working together instead of taking turns doing things. Everyone in class did something wrong or one of the reactants we all used failed to work. I feel that my pipetting became more honed. Where as adjusting pH is unchanged from the beginning of the project. I would like to practice the pH adjusting a little more. At the end of the day, all this lab did was to make me even more confused about all this DNA stuff.
My group worked decently well. We could have been more efficient by working together instead of taking turns doing things. Everyone in class did something wrong or one of the reactants we all used failed to work. I feel that my pipetting became more honed. Where as adjusting pH is unchanged from the beginning of the project. I would like to practice the pH adjusting a little more. At the end of the day, all this lab did was to make me even more confused about all this DNA stuff.