First Lab Day
First day in Lab!
-Sam White gave us an orientation of the lab and safety protocols in the morning.
-I began making some reagents for the protein extractions today:
5 ml of 200mM Dithiothreitol (DTT) reagent
I labeled 25- 1ml snaptop centrifuge tubes for the 200ul aliquots I plan to make. Used a pipette to add 5.00ml of nanopure to a 10ml falcon tube. I weighed out 0.1543g of Dithiothreitol and added to the falcon tube to dissolve with the nanopure water. Inverted several times to dissolve. Once dissolved, I aliquoted 200ul into each of the labeled centrifuge tubes and stored immediately in the -80C freezer.
5 ml of 1.5M Tris(tris(hydroxymethyl)aminomethane hydrochloride) pH8.8
I used a pipette to add 2.00ml of nanopure to a 10ml falcon tube. I weighed out 1.182g of Tris and added to the 2ml of nanopure in the falcon tube. I realized I didn’t have enough liquid in the falcon tube to dissolve it so I added another 1 ml. I used a pH meter that we calibrated prior using pH buffers 7 and 10, and found the pH of this solution to be around 4. Because we need the solution to be pH 8.8, I added a drop of 5N NaOH. This increased the pH about 2 units so I slowly added more 5N NaoH until I reached pH of 8.8. I ended up using 23 drops. At this point I was at 4.8ml verfied using a glass buret. I added 200ul more of nanopure, and checked the pH. The final pH of the solution is 8.89. I kept the solution in a labeled falcon tube on the benchtop.
Solvent A (60% acetonitrile + 0.1% trifluoroacetic acid)
The goal is to limit contact of any of these chemicals with plastic. I added 59.85 ml of nanopure water to a Nalgene glass bottle using a 50ml graduated cylinder and pipette. I then added 90ml of acetonitrile using the 50ml graduated cylinder. Finally, I poured less than 1ml of the trifluoroacetic acid into a glass testtube so I could pipette 150ul to my final solution.
Solvent B (5% acetonitrile + 0.1% trifluoroacetic acid) Again, avoid plastic! I added 142.35 ml of nanopure water to a Nalgene glass bottle using a 50ml graduated cylinder and pipette. I then added 7.5ml of acetonitrile using the 50ml graduated cylinder. Finally, with the excess trifluoroacetic acid that I had left in the testtube, I pipetted 150ul into my final solution.