Grace’s Notebook: Tuesday, November 7, 2017

Yesterday, I went with Laura to Manchester to help clean and de-symbiont about 1600 oysters. There were 7 or 8 of us working, so we got it all done in a little over three hours! And, it was thankfully a beautiful, sunny (cold) day. I scraped off tons of barnacles, limpets, algae, and red worms.

Today, I worked more on the DIA analyses protocol. Specifically, Step 2. Some issues came up, so Steven and Sam have both been helping address them so that we can move forward. I am now looking at 3a and 3b. My task is to work on understanding what these steps mean, and then Steven said we’ll wait and see what we can do about running Pecan. Apparently, it’s difficult to use, and may have to be sent to Emma to run.

I then revisited this GitHub Issue and took some images to provide an example of what this project would be like if I followed suit with what I’ve done in the past.

Sam, Steven, and I (I mostly stood watching) worked on connecting the titrator to the computer (Swan). It was complicated, but I think they got it to work. My task for tomorrow is to make the NaOH (0.1 mol/L) solution in the titrant bottle so that we can hopefully start moving forward with the titrator!

 

Grace’s Notebook: Tuesday, January 9, 2018

(1) 2015 OysterSeed Data

Today I revisited the 2015 OysterSeed skyline project. I left off at Step 5b .

I am going through ~100 peptides to check that Skyline chose the correct peaks. Peaks are there when peptide or transitions were detectable, and the greater the peak, the stronger the detection was.

I am not sure what to do when the peaks are inconsistent across all oyster seed samples within a peptide. I believe that in Yaamini’s SRM protocol, I was to delete the peptide if the pattern was inconsistent between samples.

Additionally, I am supposed to determine the per-sample and per-peptide error rates. To do this, I will be following what Yaamini did in her spreadsheet: here. Unfortunately, I asked about this after already checking 26 peptides. I didn’t keep track of which ones I checked, so I’ll have to start over tomorrow. I don’t think it should take too long.

(2) Roberts Lab Onboarding

I am going to transition to using a GitHub notebook in order to keep things to do with my projects more organized.

Grace’s Notebook: Tuesday and Wednesday, December 19th and 20th, 2017

Today and yesterday I finished spinning down, and saving the supernatant and pellet of the final samples.

I added the labeled boxes to the -80 (Tray 14).

Pam picked up the 50µl samples this morning.

Grace’s Notebook: Monday, December 18, 2017

Today I was in for a little over an hour labeling tubes in preparation for tomorrow and Wednesday. I will be spinning down the final batch of Tanner Crab hemolymph and RNAlater mixtures, and saving the supernatant and pellets in separate tubes in the -80.

Grace’s Notebook: Friday, December 15, 2017

Today I set aside 50µl of crab hemolymph and RNAlater mixture for Pam. 117 tubes for the 117 crabs that remained at the end of the project. Storing them in labeled boxes in the refrigerator in Rm 213.

Next week I will spin down all the tubes, keep the supernatant and pellets in separate tubes and store in -80. Hoping that more P1000 pipet tips will arrive by then to help speed things up.

Grace’s Notebook: Thursday, December 14, 2017

(1) 2015 oyster seed data

I went through some more peptides on SkylineDaily with the 2015 oyster seed data. Protocol calls for checking ~100 peptides to ensure correct peak was selected (Step 5).

(2) Crab project

I met with Steven and Pam today to discuss the overall project and next steps.

Pam brought the most recent samples. There are three samples per crab, and 6 per warm-water treatment crabs (because only 3 survived). Total of 117 crabs.

I will save 50µl of hemolymph/RNAlater mixture for Pam from each crab (117 tubes total). Then I will spin down all the samples (114×3 + 3×6 = 360 samples total), remove and save the supernatant, and place supernatant and pellet tubes in -80.

In January, we will start processing the RNA and pooling the samples to be sent off for sequencing.

Grace’s Notebook: December 12, 2017

Crab hemolymph samples:

Pink-capped were sampled 11/10/2017

I spun down the hemolymph samples (5000g for 10min) and saved the supernatant and pellets in separate tubes. The boxes were placed in the -80 (tray 14).

Grace’s Notebook: December 11, 2017

Crab Hemolymph Samples:

Green-capped were sampled 11/08/2017

I spun down the crab hemolymph samples and saved the supernatant and pellets in separate boxes. I spun them down at 5000g for 10 minutes. Then, I placed the boxes in the trays in the left -80. (Tray 8 and Tray 10)

I only got through the green-capped samples today.

Grace’s Notebook: Juneau, November 28, 2017

Today Pam and I cleaned and packed up the lab!

We sent the coolers, pumps, and other things to the Kodiak Lab, and sent some of her things back to Seattle.

We power washed the tanks and scrubbed them down.

Pam has to send samples with ethanol, and they can only be sent through UPS. In Juneau, UPS is only open from 7pm-8pm.

Tomorrow Pam is giving a talk around 11am about this project. We have a few more things to clean and pack up, and then we’ll be done!

Grace’s Notebook: Juneau, November 27, 2017

*I will double check some specifics of information (denoted by * or __) with Pam and edit post accordingly.

Effects of temperature change and Hematodinium sp. infection (Bitter Crab Disease) on Tanner crab (Chionoecetes bairdi)

Final sampling day of Tanner Crab (Chionoecetes bairdi) blood 

Pam Jensen and I arrived at the Ted Steven’s Marine Research Institute in Juneau at ~7:45am. We worked in the Annex (right image).

NOAA facility

Annex

Experimental Set-up

There were three header tanks at different temperatures (4˚C – cold; 10˚C – warm; ~6˚C – ambient). The cold and ambient had coolers, and the warm had heaters. The water flows (2L/min) into the tanks.

Cold (4˚C)

Warm (10˚C)

Ambient (___˚C)

There was a mass mortality in the warm tanks due to the temperature getting too high over a weekend. As a result, by today’s sampling day, there was only one crab in each of the three warm tanks.

Set-up of the lab

The header tanks with the coolers had brass floats. If the water went to high, it would close the valve with the water source (much like a toilet) to prevent the water from overflowing as well as the temperature becoming too cold. Additionally, both cooling tanks had pumps to circulate the water to homogenize the temperature.

Brass float

Cooler

Inside cooler

The temperature was logged every 15minutes using HOBO Tidbits. One TidBit in each tank (9 total).

Tanner Crab phlebotomy 

The crabs were corralled into one end of the tank, and a screen was placed to hold them back. Additionally, bricks were laid at the bottom because the crabs could crawl under the gaps in the screen. In between samples, we kept foam over the top of the tank, to keep the temperature from fluctuating too drastically, since the room was likely quite a bit warmer (even though we did not have the heat on) than the water in the tanks.

Crabs to be sampled.

Each crab has two tags of different numbers. There are two because sometimes the tags will come off, or their entire leg with the tag, will come off. All the crabs are male and young in order to keep from having too many variables.

After the tag numbers were recorded with a tube number, a Q-tip with rubbing alcohol was rubbed in order to clean the area of other dinoflagellates. Then, a syringe is used to sample the blood at the base of the front claw.

Rubbing alcohol

Sampling blood with a syringe

A drop of blood is placed on a labeled slide to create a blood smear, and then 0.2 ml is placed in three tubes containing RNAlater (6 for the warm water crabs, because we only had three total).

The blood smears will be stained by _____ at _____. She will use the slides to determine the life stage of the Hematodinium.

Using another slide, the blood would be smeared.

0.2 ml of blood into RNAlater

Clean up

The blood smear slides were let to dry and then placed in a slide box. The HOBO Tidbit temperature data were downloaded.

Pam and I began the process of taking down the lab. We had to sacrifice all the crabs even though they were collected from Stevens Passage in Juneau. Pam removed the tags and placed them in the dumpster.

We took apart all the plumbing and pumps. Rinsed everything with freshwater, drained the tanks.

Tomorrow we’ll clean, pack everything up, and ship things to the Kodiak Lab.

Other Info:

Pam said that while other types of infection can cause this mottled appearance in their legs, it is likely the result of Hematodinium infection because ~50% of Tanner Crabs are infected in the fall in  the Juneau area.