Katie’s Notebook: 6/1/17

Took Laura’s K-treatment oysters to UW today, and discussion started about what projects I might be interested in doing this summer. We put the oysters in separate containers by temperature and OA treatment in warm salt water after being in a cooler all night to see if they would open up or spawn. We saw some activity, but are going to leave them overnight and then Steven will separate them all out into individual beakers tomorrow morning. Hopefully we get lucky and some were brooding and we see some larvae! We are hoping to do some experimenting with how I could best track spawning time and larval release.

I also spent some time researching small cameras that are used to record activity underwater and maybe even inside the oyster. Endoscopes are what have been used most often on bivalves. Specifically with investigating food movement throughout the gills and just general movement of particles taken in from the water. They come in a variety of sizes/quality.

Sean’s Notebook: Things went from…

Sean’s Notebook: Things went from not working, to working. It’s a miracle!

So I’d been having problems with Platanus throwing a segmentation fault during graph construction with a smaller k-mer length argument, which was a pain because it’s such a generic error. Turns out it just needed more memory, because 400gb isn’t enough? Hopefully it will be done by tomorrow and we can get try Redundans again, with better results!

Also, I got MethylExtract working on the C. virginica methylation data. Looks like when you have PBAT data, you have to switch the flags to flagW=0 and flagC=16, even though you’re using paired end data.

MethylExtract notebook: here
C. virginia CG and SNP data: here pending renaming/reorganization of files.

Sean’s Notebook: All TA, all the time.

Spent another day at DNR in Olympia running water samples. Hollie suggested I try drawing acid directly from the stock container to remove the chance of changing acid concentration by mixing old acid and new acid in the titrator reservoir. I did this, as well as mixing the acid bottle before each sample run and still found a frustrating amount of variation in CRM samples. Micah was available to watch my technique and we decided it did not appear to be caused by operator error.

We re-checked the probe calibration and found that it had drifted 5mV at 3.5pH in the week since it was last checked, so we adjusted the titrator script and ran a set of CRMs in the middle of the day and found better consistency with results, but they were still reading higher than intended. We decided that in the future checking the probe calibration at the beginning of each processing day would be a good move.

After thinking of other potential reasons for consistently high CRM readings, we checked the calibration of the balance used to weigh out samples, thinking a higher than expected mass reading could lead to higher than expected TA readings, but after finding a reference weight in the nearby geology lab, we found that the scale was accurate within 0.01g, so that wasn’t the culprit. The only other thing we could think of, was potentially the acid burette was dispensing an incorrect volume of acid. Micah said that he would contact Mettler-Toledo regarding potential ways to test this/ways to calibrate it, and will go from there!

TA values: here
Raw data: here

Katie’s Notebook: Manchester Week 2

After learning the full routine of screening, sampling, counting, restocking, and then collecting all the extra larvae to be frozen last week, things went much more quickly today. Laura was doing a lot of rearranging to make sure all her larval buckets are getting the same quality of food and are under the same conditions. I collected any new larvae that had spawned to screen through 100um and sample for counts. We got all the rearranging and restocking done before lunch!

After lunch Laura and I discussed possible research projects for me to work on this summer. Some of our ideas included:
-Histology + larval counts and how long does one oyster spawn
-Respiration experiment, metabolic difference
-Observing new larvae size differences from different treatments
-O2 trials?
-Mechanical stress or feeding experiment + mortality rate

We were very interested in discussing the project concerning the observation of the new larvae. Possibly using imageJ to track size differences and growth. This would be interesting because this work has been done on Pacific oysters, but not Olympic oysters who brood their young. It also would contribute to Laura’s project as well.

I was sent home with 15 oysters from each of the 4 treatments from the K-group that I will take to UW on 6/1 in case I want to use those for my project.

Yaamini’s Notebook: June Goals

(Almost) done with Spring Quarter already?!

Can’t believe it’s already June! In keeping with the April and May #MemeThemes, here’s a picture of Mama June from Here Comes Honey Boo Boo that properly expresses my reaction to the passage of time.

1q3ddg

May Goals Recap

  • Developed a preliminary heat shock plan, but it needs to be revised
  • In-depth spawning plan preparation put on halt because I can’t get in contact with Molly
  • Skyline is tempermental so I don’t have a list of targets yet
  • I wrote an introduction for the DNR paper! It’s rough, but it’s made of real words 🙂
  • Not only do I have an extraction plan, but the extractions are well underway
  • I have the pipeline for my metaanalysis ready to go, but I need either emu or roadrunner to be free so I can start. The days those machines are free are when I’m not, so by the time I’m free, the machines are occupied again. Whoops :stuck_out_tongue_closed_eyes:

June Goals

  • Heat shock oysters
  • Have an in-depth spawning plan
  • Get Skyline to function
  • Create a list of targets
  • Finish extractions
  • Finally, FINALLY start that metaanalysis pipeline so it’s no longer a pipe dream

It’s quite a chunk of work, especially considering I’ll be working from California for a week. But I think it’s doable considering the fact that I won’t have any classes!

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Yaamini’s Notebook: DNR Mini Trypsin Digestion Round 3

#

Six boxes of trypsin arrived yesterday. Guess what that means?!

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Quick tangent: my schedule shifted around a bit, so here’s an updated extraction schedule. I want to finish all of my extraction work before I work from California for a week.

Thursday (today!): Mini Trypsin Digestion – Jose and Yaamini (ft. Kaitlyn) Friday: I speed vacuum my samples. This way, I can use the weekend to study for my QSCI final and wrangle with Skyline and MSstats. Monday: Desalt all my samples. Forget about all labwork until mid-July. Spend countless hours at Manchester and at the Windows machine instead.

Here’s how Jose and I digested 100 µg of protein from each sample:

Step 1: Set up heating blocks

  • Desired temperature is 37 ºC
  • Use additional thermometers to confirm temperature

Step 2: Label new tubes for digestion

Step 3: Make 50 mM NH4HCO3 + 6M urea solution

Step 4: Pipet volume of sonicated sample into new tubes

  • Based on yesterday’s BCA Assay, I calculated the volume of sonicated smaple necessary for 100 µg of protein

Step 5: Pipet volume of 50 mM NH4HCO3 + 6M urea solution into new tubes

  • Combined, the sonicated sample and 50 mM NH4HCO3 + 6M urea solution volume in the new tubes should be 100 µL

Step 6: TCEP Incubation

  • Add 6.6 µL of 1.5 M Tris pH 8.8 to each sample
  • Add 2.2 200 mM TCEP to each sample
  • Vortex gently
  • Check pH of solution to ensure it’s still basic
    • Pipet 2 µL of solution onto the green square of a pH strip. If the color is blue, it’s basic.
  • Place samples on heating block
  • Incubate for one hour at 37 ºC

Step 7: IAA Incubation

  • Obtain IAA from the freezer. Cover with aluminum foil.
  • Add 200 µL IAA to each sample
  • Vortex gently
  • Cover samples with aluminum foil
  • Incubate for one hour at room temperature in the dark

Step 8: DTT Incubation

  • Add 200 µL DTT to each sample
  • Vortex gently
  • Incubate for one hour at room temperature

Step 9: Lys-C Incubation

  • Add 1.65 µL Lys-C to each sample
  • Vortex genly
  • Incubate for one hour at room temperature

Step 10: 25 mM NH4HCO3 and Methanol Addition

  • Prepare 25 mM NH4HCO3
    • Need 800 µL for each sample
    • 100*800 = 80000 µL = 80 mL 25 mM NH4HCO3 needed
  • Add 800 µL NH4HCO3 to each sample
  • Obtain HPLC grade methanol from flammable storage
  • Pour out necessary methanol into a clean beaker
    • Need 200 µL for each sample
    • 100*200 = 20000 µL = 20 mL methanol needed
  • Place methanol in fume hood
  • In the fume food, add 200 µL methanol to each sample
  • Vortex samples gently

Step 11: Trypsin Digestion

  • Obtain 20 µg Trypsin bottles from fridge
  • When ready to use Trypsin, add 20 µL nanopure water to the bottle
    • Creates a 1 µg/µL solution
  • Add 3.3 µL trypsin to each sample
    • One bottle provides for 6 samples
    • Need about 15 bottles for all samples
  • Vortex samples gently
  • Let samples sit overnight
    • Digestion start:

Step 12: Stop Digestion

  • Place all samples in the -80ºC freezer
    • Digestion end:

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