LAB 4A DNA! BTW this lab is 4 labs put together...
Materials
Analytical balance
Tabeltop milligram Balance Lab scoops Sodium Chloride 15mL Capped tubes Tube racks Edta TRIS Disodium Salt 125 ml bottle Glasses Graduated Cylinder |
Sodium Hydroxide
Hydrochloric acid Glass Rods 50 ml beakers 2 ml pipette Salmon sperm DNA P1000 micropipette Ethanol, 95% Permanent markers 1ml plastic peaker TAE buffer concentrate, 40X 600ml beaker Agarose |
Microwave
Hand protecters, heat Gel box Water bath Reaction tubes, 1.5ml DNA samples Loading dye Micro-pipettes Media bottle, 250ml Microcentrifuge Ethidium Bromide Gloves UV light |
Important Concepts:
Conversions from Liters to Moles: 10 mL= 0.01 L, 10 mM= 0.01 M
Precipitation- taking something out of the solution. The precipitate doesn't dissolve in a solution
DNases- enzymes that break down DNA
NaCl- sodium ions are positively charged. Sodium reacts with the phosphate in DNA molecules it makes it clumpy.
TRIS- keeps pH of solution neutral because if the solution is too basic or acidic, the molecules will come apart, which will mess everything up.
EDTA- A preservative that prevents DNase activity
Molarity Calculation: (Molarity)(Volume)(Formula Weight) = g substance needed
Other Calculation who's name I forget: (Stock solution concentration)(Volume of stock solution to use) =(Final desired concentraion)(Final desired volume)
Conversions from Liters to Moles: 10 mL= 0.01 L, 10 mM= 0.01 M
Precipitation- taking something out of the solution. The precipitate doesn't dissolve in a solution
DNases- enzymes that break down DNA
NaCl- sodium ions are positively charged. Sodium reacts with the phosphate in DNA molecules it makes it clumpy.
TRIS- keeps pH of solution neutral because if the solution is too basic or acidic, the molecules will come apart, which will mess everything up.
EDTA- A preservative that prevents DNase activity
Molarity Calculation: (Molarity)(Volume)(Formula Weight) = g substance needed
Other Calculation who's name I forget: (Stock solution concentration)(Volume of stock solution to use) =(Final desired concentraion)(Final desired volume)
Purpose 4A +4B: The purpose of this lab was to create two solutions by correctly calculating amounts of specific substances to make proper solutions. The two solutions we made were 10 milliliters of 5 mole NaCl solution and 100 milliliters of 10 micro moles of TRIS and 1 micro mole of EDTA.
Procedure:
1. Make molarity calculations.
1st Solution NaCl: 5M x 0.010 L x 58.44 g/mole = 2.92 grams
2nd Solution TRIS: 0.01 M x 0.1 L x 372.24 g/mole = 0.158 grams
3rd Solution: 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.
1st Solution NaCl: 5M x 0.010 L x 58.44 g/mole = 2.92 grams
2nd Solution TRIS: 0.01 M x 0.1 L x 372.24 g/mole = 0.158 grams
3rd Solution: 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.
Purpose 4i: The Purpose of this lab was to make the agarose gels to run our salmon DNA through it.
Procedure:
1. Prepare solution of 0.8% agarose in 1X TAE buffer solution.
2. Weigh out required powdered agarose in a weigh boat. Place in 250-mL media bottle.
3. Measure out TAE to prepare 100 mL of agarose and buffer mixed together.
4. Dissolve agarose in microwave by bringing to boil.
5. Let cool until cool enough to hold.
6.Pour the gel into a gel box.
7. Put comb into the notches in the gel tray.
8. Pour 1X TAE buffer onto the gel box until covering the gel.
9.Wrap in Saran wrap and store.
1. Prepare solution of 0.8% agarose in 1X TAE buffer solution.
2. Weigh out required powdered agarose in a weigh boat. Place in 250-mL media bottle.
3. Measure out TAE to prepare 100 mL of agarose and buffer mixed together.
4. Dissolve agarose in microwave by bringing to boil.
5. Let cool until cool enough to hold.
6.Pour the gel into a gel box.
7. Put comb into the notches in the gel tray.
8. Pour 1X TAE buffer onto the gel box until covering the gel.
9.Wrap in Saran wrap and store.
Purpose 4J: The purpose of this lab was to test the salmon DNA in electrophoresis.
Procedure:
1. Put 20 micro liters of salmon sperm DNA and 3 micro liters of DNA loading dye in 1.7 mL tubes. Spin tubes in a minicentrifuge.
2. Place salmon DNA and dye mixture into wells of gel using micro pipette.
3. Connect the electrodes of the gel box to the power supply and run the gel.
4. Run until you see dye move halfway down the gel.
5. Cover the gel with EtBr (ethinium bromide) for 20 minutes. Pour off the EtBr, Pour on deionized water, and observe the gel on a UV light box.
1. Put 20 micro liters of salmon sperm DNA and 3 micro liters of DNA loading dye in 1.7 mL tubes. Spin tubes in a minicentrifuge.
2. Place salmon DNA and dye mixture into wells of gel using micro pipette.
3. Connect the electrodes of the gel box to the power supply and run the gel.
4. Run until you see dye move halfway down the gel.
5. Cover the gel with EtBr (ethinium bromide) for 20 minutes. Pour off the EtBr, Pour on deionized water, and observe the gel on a UV light box.
Conclusion/Analysis:
Sadly our lab didn't work, but we learned a lot so it's all good. We couldn't see the DNA activity, which could have happened because of a couple reasons:
1. 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.
We could test this by...
Making a new batch of stain: 20,000x stock - diluted to 1x
OR Stain gel with new solution for three hours.
2. We did not mix the sperm enough, meaning we didn't get DNA in our sample.
- This is extremely unlikely because all the gels did not run the DNA, and everybody messing up is like winning the lottery.
3. 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.
4. 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.
5. One of the reagents went bad.
- This is possible because the stain is light sensitive, and could have broken down.
6. We did not make our solutions correctly.
Sadly our lab didn't work, but we learned a lot so it's all good. We couldn't see the DNA activity, which could have happened because of a couple reasons:
1. 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.
We could test this by...
Making a new batch of stain: 20,000x stock - diluted to 1x
OR Stain gel with new solution for three hours.
2. We did not mix the sperm enough, meaning we didn't get DNA in our sample.
- This is extremely unlikely because all the gels did not run the DNA, and everybody messing up is like winning the lottery.
3. 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.
4. 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.
5. One of the reagents went bad.
- This is possible because the stain is light sensitive, and could have broken down.
6. We did not make our solutions correctly.
Reflection: My group worked okay together. We made some mistakes that were very preventable. For example, when we were making our solution neutral we made it way way way to acidic. This was preventable because all we had to do was add a couple drops but we squirted it in. Other than a few hiccups our project went smoothly and it was very fun. Next time I might try a different group to do the extreme accuracy parts of the lab just to makes sure it all goes well.