DNR Mini-Trypsin Digestion Round 2
I need to become a faster pipetter.
If there were a pipetting Olympics, I wouldn’t come in last. But I wouldn’t come in first either, or even medal. This was slightly problematic when pipetting every few minutes for the trypsin digestion. It took a while, but we finished!
Last time we performed a digestion, our quantities were calculated based on having 30 µg protein/100 µL. This time, we had 100 µg protein/100 µL. Therefore, we used the quantities of reagents specified in the original protocol.
The first thing Jose and I did were set up heating blocks at 37ºC. Because the heating block in the Roberts Lab did not have the capacity to hold all of our samples, Jose brought one from his lab. We placed additional thermometers in the heating blocks to ensure the temperature was what we wanted.
Based on my calculations for Plate 1 and Plate 2, I pipetted the correct amount of sample and 50mM NH4HCO3 + 6M urea into newly labelled tubes (ex. OBLNK D). Then, I added 6.6 µL of 1.5 M Tris pH 8.8 and 2.2 200 mM TCEP and vortexed the tubes. For our 51 samples, we needed 2-200 µL aliquots of TCEP from the -80ºC freezer. At this point, I went to class. Jose tested the pH of our samples to ensure the pH was still basic, than began the digestion in the heating blocks for one hour.
Figure 1. pH strips for samples. The blue color of the strips indicates the sample pH was still basic.
Figure 2. First heating block set-up.
Figure 3. Temperature confirmation for first heating block.
Figure 4. Second heating block set-up.
Figure 5. Temperature confirmation for second heating block.
After the digestion, I got 6-200 µL of IAA from the -80ºC freezer and covered them with aluminum foil. We added 20µL of IAA to each sample and vortexed the tubes. We covered all of our samples with aluminum foil and let them digest for one hour. Halfway through the digestion set-up, I left for Merril’s Ph.D defense. When I was gone, Jose added 20 µL of DTT to each of our samples, vortexed, and started the digestion. I got back in time to add 1.65 µL of LysC to each sample and started the one hour digestion.
Because Jose needed to leave early, he prepared 25 mM NH4HCO3 solution for all of our samples. After the digestion was over, I added 800 µL of 25 mM NH4HCO3 and 200 µL of HPLC grade methanol to each sample and vortexed them. I then needed to add 3.3 µL of Trypsin to each sample, meaning I needed 9 bottles of 20 µg Trypsin. We only had five bottles, so I needed to borrow 4 bottles from Genome Sciences.
Before using each bottle, I added 20 µL of nanopure water and vortexed. This made a 20 µg/20 µL solution, allowing me to add 3.3 µL of Trypsin to six samples/bottle. After adding Tryspin to all oyster and geoduck samples, I vortexed the tubes and let them sit overnight. I started the digestion at 4 p.m. on Friday evening, and stopped it at 10 a.m. Saturday morning.