Purpose:
The purpose of this lab was to learn how to make solutions and use our solutions to be able to observe and learn more about DNA.
MAterials:
Balance (Analytical and Tabletop)
Weigh Paper Weigh Boats Lab Scoops Sodium Chloride Tubes (With Racks) TRIS EDTA Ethanol (95%) 600mL Beakers |
125 mL Bottle
100 mL Graduated Cylinder pH Paper Hydrochloric Acid Sodium Hydroxide Glass Rods 50 mL Beakers DNA (Salmon Testes) 2 mL Pipet (With Pumps) |
Agarose
Microwave Oven Hot Hands Protector Safety Goggles Gel Box Power Supply Centrifuge Ethidium Bromide Micropipet Tips (With Pump) |
Procedure:
Lab 4a:
Part I: Preparing 5M of NaCL
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 II: Preparing TE Buffer
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
Part I: Preparing 5M of NaCL
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 II: Preparing TE Buffer
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 (forms two layers)
4. Using a glass rod, spool DNA
5. Place DNA in a tube with 2 mL TE
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 (forms two layers)
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 (with combs)
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 (with combs)
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
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:
Unlike our other experiments this year, this lab did not work. After staining with Ethidium Bromide, no DNA showed up. This was universal--not just our group. Therefore we can conclude that the most likely issue was our loading dye expiring. To test this, a new batch of loading dye as well as a new stock batch of loading dye will be made and tried again. Hopefully our experiment can be salvaged!
UPDATE:
After making a fresh batch of gel and restaining, we were able to see the gels. (see pictures below)
After making a fresh batch of gel and restaining, we were able to see the gels. (see pictures below)
Conclusion:
In this lab, we tried (and failed) to separate DNA from a solution onto a gel. The value of doing this is the hands-on experience in the lab, the ability to actually see DNA, and to reinforce our knowledge about the polarity of DNA. These techniques could be used in a biotech of forensics lab to study DNA or even to identify a culprit in a crime.
Reflection:
As a group, we did not work very well. A few members barely did anything while others did more of the work. Just as in our other projects, we have not shared our work burden evenly and we have to work on better division of work. We could have been much more efficient if we would have collaborated more instead of messed around. We must have made mistakes (as evident in our lack of DNA) and will probably have to redo that lab as a result. In the future, I will try to thoroughly understand everything before executing any step that could possibly affect the result. My pipetting skill has grown and I feel confident in my abilities. However, I am not as confident about making solutions and adjusting pH. I could use more work on that. Overall the project felt very shallow and I don't feel as though I've learned anything. However that may be due to our experiment failing. In the future I will try to work more diligently to learn better.