11/21/2016

Preliminary Steps

I pulled the 22 samples we will run out of the main box and put them in a seperate labeled box in the -80C freezer.

Labeled 3 new sets of the 22 snaptop centrifuge tubes with the number assigned to each sample we will run.

First thing in the morning I made 20ml of the 50mM NH4HCO3 in 6M Urea. To do so I added 10ml of nanopure using a glass graduated cylinder into a 45ml falcon tube. Then I weighed out 79.06mg of NH4HCO3 and added to the falcon tube and vortexed to dissolve. Next I weighed out 7.21g of Urea and added this to the falcon tube. Again vortexed to dissolve. Then I topped off the volume to 20ml by pouring the solution back into the graduated cylinder and adding nanopure. Poured solution back into falcon tube.

Brought a cooler and got dry-ice from building near Bagley. Needed SAFS box number 355020 and Roberts budget number.

Sonication Process

1) Obtained the 22 samples out of box in the -80C and placed in rack. I tried our sonicator 11/21/16 and it broke. I went to the Genome Sciences building to use theirs on 11/22/16.

2) Added 500ul of the 50mM NH4HCO3 + 6M urea solution to each of the 22 samples. Worked in numerical order to make sure the timing of each of these steps was as similar as possible for each sample. Homogenized each sample with a clean blue plastic pestel. Since the cryotubes didn’t fit in the centrifuge, I pipetted the supernatant into a clean labeled snaptop centrifuge tube. I placed them in the centrifuge set at 2000rpm for 5 minutes.

3) After the centrifuge step, I pipetted out 150ul of supernatant liquid from each sample and put into clean labeled tube. Saved the remaining samples in the -80C.

4) Brought samples, wet ice, and dry-ice over to Genome Sciences Building.

5) Made an ethanol dry ice bath. Used a plastic container and added ethanol first. Then I carefully dropped in pieces of dry ice. Once the dry-ice was not dissolving very fast, the solution was cold enough.

6) Made a wetbath. Got ice and placed in cooler.

7) Sonicated first sample for 5s (setting is at 5) then dipped in ethanol/dry ice bath for 5s. Repeated this process twice for a total of 3 sonication rounds. Once finished, I placed the sonicated sample in wet bath (just wedged samples directly in ice). Cleaned sonication tip with ethanol and wiped with kim wipe. Repeated this process for the rest of the samples.

8) Brought samples back to FISH209.

9) For each sonicated sample, I pipetted 11ul into a new labeled centrifuge tube for protein quantification. I now have 4 tubes for every sample (original cryotube with homogenized larvae, one snaptop centrifuge tube with leftover unsonicated sample, one snaptop centrifuge tube with 11ul for protein quantification, and one snaptop centrifuge tube with remaining sonicated sample for protein digestion).

10) Placed all of these samples back in the -80C.