Lab 4a & 4b
Hypothesis: The DNA will create a clear band that we can see and be able to use later.
Purpose:
1. To make 10ml of 5M NaCl solution
2. To make 100ml of TE Buffer
3. Use the 2 solutions to spool together DNA and put into a test tube
Purpose:
1. To make 10ml of 5M NaCl solution
2. To make 100ml of TE Buffer
3. Use the 2 solutions to spool together DNA and put into a test tube
Materials:
- Analytical Balance
- Tabletop milligram balance
- Weigh paper
- Lab scoops
- Sodium chloride
- 15ml tubes
- Tube racks
- TRIS
- EDTA disodium salt
- 125ml bottle
- 100ml graduated cylinder
- pH paper
- Hydrochloric acid
- Sodium hydroxide
- Glass rods
- 250ml filtering flasks
- 50ml beakers
- Salmon testes DNA
- 2ml pipet
- P-1000 micropipet + tips
- Ethanol 95%
What is Precipitation?
- In Science, precipitation is taking something out of a solution
What are DNases?
- An enzyme that breaks down DNA
Why use NaCl?
- Charge of the sodium binds to DNA so it clumps together to form a precipitate
Why TRIS?
- It maintains a good pH of about 7-8
Why EDTA?
- It prevents DNase activity during the lab
- In Science, precipitation is taking something out of a solution
What are DNases?
- An enzyme that breaks down DNA
Why use NaCl?
- Charge of the sodium binds to DNA so it clumps together to form a precipitate
Why TRIS?
- It maintains a good pH of about 7-8
Why EDTA?
- It prevents DNase activity during the lab
Lab 4a:
Solution 1, 5M NaCl:
Solution 1, 5M NaCl:
- First we had to determine the amount of NaCl needed. We use the formula M(Mole concentration) * V(Volume) * FW(Formula Weight). Doing this we get (5M)(0.010L)(58.44) = 2.92g NaCl
- Next we used the analytical scale to carefully measure 2.92 grams using he weigh paper.
- We then put that into a test tube and poured de-ionized water up to 15ml in the test tube, mixing them with the vortex machine.
- Determine the amount of TRIS and EDTA for TE Buffer using formula M * V * FW
- TRIS: 0.010M * 0.100L * 157.6 grams/Mole = 0.1576 grams TRIS
- EDTA: 0.100M * 0.1L * 372.24 g/M = 0.372 grams
- Being even more careful and precise than last time we used the analytical scale to get the amounts and put them into the same 125ml tube. Then we filled the tube up to 100ml and mixed them well.
Lab 4b:
- Dilute the DNA with the TE Buffer in the beaker. Observe the TE and the salt water and DNA together.
- Add the NaCl and observe all together. You can at this point see layers forming between the DNA and the Buffer
- Add 4ml EtOH(Ethanol) down the side of the beaker to not disturb the DNA.
- Use the glass rods to spool the DNA together
- Put the clumped DNA into a new test tube with 2ml of fresh TE buffer and label
Lab 4i
1x TAE Buffer
Use C1V1 = C2V2 C1 = Stock Concentration V1 = Volume stock we need C2 = Final Concentration V2 = Final Volume V1 = (1)(500)/ 40 V1 = 12.5 ml 40x TAE + 487.5 ml water |
50ml 8% agarose in 1x TAE
Need 8% of 50ml = .008 X 50 = 0.4 agarose Add with 50 ml TAE Buffer in erlenmeyer flask |
Purpose: To makes a TAE Buffer for the DNA and agarose for the gel for the DNA
- Make 500ml 1x TAE Buffer and 50ml 8% agarose
- Add agarose to 100ml 1x TAE in 250ml flask
- Heat and swirl to boil and dissolve
- Let flask cool until you can touch it
- Get gel container and put tape all around each side to hold in the agarose
- place in 2 combs in the 2 slots of the container
- When ready pour in the mixture to the gel box and let cool
Lab 4j
Purpose: To use the gel and DNA to create a DNA band to study
- Remove tape from gel, place in gel tank
- Pour TAE over gel until covered, gently remove combs
- Prepare Samples: 20 ml DNA + 4ml 6x loading dye using the micropipet from 2-20; spin for 2 seconds together in mini centrifuge
- Load samples onto gel using 20-200 micropipet
- Cover gel tank, plug into power supply
- Run at 110V for 45 minutes
- Stain several hours with EtBr
Data Analysis
After the whole lab we did not actually see any DNA bands, so my hypothesis was incorrect. Instead, the gel appeared clear with purple dots and they were all about the same distance from the beginning position before the electricity ran though them. There were seven dots for each person. There could be many reasons for the lab to have not produced a DNA band. Most likely though, the EtBr dye probably corroded during the process so we can't see the band, even though it is likely there. We did end up running the gels over again and it turned out better this time. The dye stayed and the DNA was highlighted in a lighter area in each strip. We could now clearly see that the DNA had moved.
Conclusion
There is value in doing a lab like this for scientists. I can see how this could be useful to isolate the DNA and then further study the DNA. This would likely be used in a forensics lab to be studied for analyzing who the DNA is from or possibly some other biotech situation to modify DNA with improvements.
Reflection
For the lab I found our group worked pretty well together. Some of our group won't take initiative for certain parts of the lab, which made it take longer since we took longer to do the parts. If more of us were doing separate things at the same time, we could have been more efficient. We probably made a few mistakes as far as making the correct concentration and amount of solutions that may have been a large effect by the end. We should take more time for measuring things so we can be more accurate in our solutions in the future. Our group did do well, I thought, at using each pipet correctly and accurately for the lab. Altogether we did well for the lab but like anything else, practice would only increase our skills even more next time.