Laura’s Notebook: Choosing the right ones.

Geoduck sample selection for next round of MS/MS

This is what I have to work with:

image

Steven, Yaamini & I decided to focus on the DNR Trial #1 for the next round of sample extraction/protein analysis.

To narrow down my sample selection further, I see that the geoduck did not fare well at the Skokomish site’s bare treatment (likely due to predation), as only 2 specimens survived to be sampled, AND Micah said that the eelgrass bed @ that site was in a freshwater seep, and therefore the salinity was significantly different there. I am therefore not moving forward with the SK site

I will select 6 samples from each site/treatment for this next round, ~2 per enclosure. To do so, I used a random number generator to select 2 to 3 replicates from each enclosure.

image

image

The file with my list of selected samples is located in my DNR Repo

from The Shell Game http://ift.tt/2qeTOVP
via IFTTT

Laura’s Notebook: May 21st 2017

Arrival Inspection

  • Aeration @ larval table not working; turned airstones up and that worked, so somehow the airline on the larval table lost some pressure overnight.
  • Valves 6, 7 & 8 on the bottom shelf’s manifold the larval table consistently bubbling from air in the FSW line, other buckets don’t have the same bubbling.
  • Banjos moderately dirty, not clogged though
  • K-10 amb larval dripper clocked, bucket only 1/2 full
  • Larvae present in catchment buckets:
    • K-6 amb: a bit
    • SN-6 low B: a lot
    • SN-6 low A: a lot
    • SN-10 amb A: a lot
    • SN-10 amb B: a lot
    • NF-10 low B: a lot

Tasks Today:

  • Measured volume actually in 5 gal bucket with banjos: 15 L = 4 gallons
  • Stocking density (as per PSRF / FAO manual): 1 larva/mL for 1 tank turnover/day
    • (24 hrs / (Tank Vol (L) / (flow rate)))1 larvae/mLTank Vol (L) * 1000mL/L = # larvae/mL to stock in bucket
    • e.g. (24hrs/(15L/8 L/hr))*1 larva/mL * 15 L * 1000 mL/L = 192,000 larvae. I’m going to round up to 200,000 larvae
  • Imaged larvae collected 5/19 & 5/20
  • Counted larvae collected 5/20
  • Plumbed in new algae input for broodstock.
    • I have noticed that the water color on the top shelf on the broodstock table is light. I’m thinking that, because the algae injection point is very close to where the pipe splits, the algae could be “channeling” to the bottom shelf.
    • Cut the 1/2 pipe ~3 feet back from the current injection point; glued in new 1/2” threaded coupler. Let dry for ~1 hour. Flushed for ~45 minutes post gluing.
  • The same issue may be happening on the larval table; there isn’t pipe available to plum in new injection point. Researched mixing valve/connection for larval line, but I didn’t find one. Need to chat with Stuart. Could just use Y’s to only use 4 middle valves and split them
  • Cleaned all banjos
  • Fed with live algae:
    • Used 1/2 Tiso/Ciso mix + CGW
  • Completed top shelf of larval table: g
    • Got new banjos finished and installed on the top shelf of the larval table
  • Replaced drippers on all lines
  • Replaced air stones as needed
  • Screened new larvae, sampled for counting & imaging
    • Screened new larvae through 200um screen onto sorting table filled w/ FSW
    • Placed 2x100um screens under sorting table- top one clean and vortexed
    • Drained sorting table onto 100um screen
    • Soaked larvae in freshwater @ 18degC for ~1 minute.
    • Collected larvae in tripour filled to 800mL for dense amounts, and to 250mL for small amounts. Labeled with tape, left on bench while I collected the other groups
    • Vortexed sorting table & screens between groups
    • Sampled triplicates from each group; counted 1 well only for stocking purposes. Fixed all wells with Lugols for counting tomorrow.
  • Stocked larval buckets:
    • Some groups that were already fully stocked on the bottom shelf spawned today, so I decided to use the top shelf of larval rearing table. This Tuesday when I screen/size/count I can either 1) separate out 2 size classes (e.g. <160um, >160um) and add new larvae to the small size class bucket until full, or 2) combine larvae from both buckets, save 200k max for one bucket, then use the empty bucket for new round of larvae. This will depend on how the larvae grow and the mortality.
    • Sketched out a method to stock buckets gradually.
      • As per PSRF, only clean/screen larvae 2x per week to minimize handling, e.g. on Tuesdays/Fridays: screen on Tuesday to size & count; then use the following calculation:
        • (200,000 – # larvae stocked on Tuesday) / 3 days until Friday = # larvae to stock on Tuesday afternoon, Wed, & Thurs.
        • E.g. stock ~50k per day on Fri, Sat, Sun & Mon for a total of 200k, then on Tuesday screen, count, and take stock of what’s left.
    • To measure out volume needed from each group, I used plunger to mix while simultaneously pouring into separate tripour to desired vol.

To Do tomorrow:

  • Measure dripper rate @ larval tank.
  • Make connection for freshwater
  • Collect new larvae, sample, count, stock
  • Determine how I should collect larval samples, and how frequently
  • Determine if I should fix larval samples in formalin moving forward as opposed to Lugols. Lugols dyes the larvae dark, so it’s not possible to decipher between live & dead. It’s helpful for counting & (I would imagine) imaging purposes, though. Ask Rhonda/Grace what was used on gigas larvae.

from The Shell Game http://ift.tt/2qLy08m
via IFTTT

Laura’s Notebook: May 18th 2017

Arrived @ 9:30am

  • lab meeting
    • can steal 2 HOBOs from gigas, leave 1
    • Brent suggested that it will take ~24 hrs of dual-bucket system to successfully collect only live larvae. This is a good idea, however I don’t expect there to be sufficient room – but maybe, if larval collections are staggered… ?
    • NEED TO: purchase more 5-gal buckets!
  • Imaged larvae collected on 5/17, and also imaged larvae collected and saved over past week.
    • Images include triplicate samples from each collection/group (see larval collection data sheet): 1x, 2x, 3x of each on Nikon SMZ645
    • Will save to GoogleDrive, then to GitHub
  • Checked catchment buckets
    • SN-10 amb B: little bit
    • NF-10 amb B: teensy amount
    • SN-10 low B: a bit
    • SN-6 amb B: little bit
    • K-10 low: some!
    • HL-10 low: maybe, probably just spermy though
    • All except HL-10 have spawned within the past 4 days, and since there is no significant amount of larvae here I will not keep them- I have a suspicion that these are from the same fertilization event, and they are trickling out of the female. Will collect for real tomorrow…
  • Stole 2 HOBOs from gigas (left 1 there); downloaded the data from the HOBOs and emailed to Yaamini, then reprogrammed and launched to record T every 15 minutes, installed in broodstock buckets.
  • Counted larvae collected on 5/17 & imaged larvae for size analysis later
  • PSRF has agreed to collect/sample my larvae tomorrow (Friday, 5/19) while I’m on campus. I pre-labeled larval rearing buckets, got water flowing, and left instructions with Jade & Alice.
  • Met with Alice to get a summary of her larval husbandry practices. PSRF hasn’t updated theirs in a couple years, and there are many lessons learned. See write-up in my GitHub (I will update as needed).
  • Screened and counted larvae, imaged. Larvae went down drain today.
  • Temperature is holding around 17.5

snip20170519_66

from The Shell Game http://ift.tt/2qLy08m
via IFTTT

Laura’s Notebook: May 17th 2017

It’s been 14 full days since we moved broodstock to their separate “chambers.” From the literature, oysters release larvae on average 10-12 days after fertilization. Today I will clean all broodstock, larval catchment buckets, after which I will plan to collect larvae to save/rear.

  • Arrived 9:30am
  • Installed 2nd immersion heater (500 watt) that I borrowed from Jon and set to 19degC. Changed set point for the other immersion heater to 19degC. Water temp seems to cool down a bit from the header tank b/c room temperature gets below 18degC at times (at night, when PSRF is trying to spawn abalone/sea cucumbers)
  • Dumped and cleaned larval buckets on east/larval table: I had left water flowing through this system overnight to test it and rinse it.
  • Cut new outflow tube for broodstock buckets- the old ones were dirty
  • Tested method of collecting only live larvae. Set up a small larval catchment bucket with banjo, removed banjo from 5-gallon larvae bucket, and increased flow using the 26 L/Hr dripper. Tested this on the SN-10 low pH B group since there were lots of larvae, and I kept an eye on things over the day. After 6 hours, I could see larvae that remained on the bottom of the bucket, which I collected and there were still lots and lots of live ones (see video). I was surprised!
  • Deployed 8 new HOBO loggers, one for each broodstock tank
  • Cleaned gigas – NOTE: no more stringy stuff!
  • Cleaned Oly broodstock:
    • Stopped flow to bottom shelf
    • Collected larvae in catchment buckets
    • Sampled larvae and fixed in Lugols
    • Collected larvae that remained in 5-gal bucket
    • Rinsed broodstock with freshwater
    • Cleaned all buckets with Vortex solution
    • Moved broodstock to other side of table, placed them randomly along manifold.
  • Screened and sampled larvae that I had been keeping alive so I could take images of them, and perhaps also to count. Dumped them down the drain!
  • I will upload and tag all images, and save in GitHub/Google Drive. Link to image location TBD.

from The Shell Game http://ift.tt/2qLy08m
via IFTTT

Yaamini’s Notebook: Another Skyline Fail

The DIA pipeline has been quite a #strugglebus

ride-this-virtual-struggle-bus-and-watch-your-lif-1-11214-1380570808-0_big

It’s unfortunate that the strugglebus website no longer works :/ Let’s go through our current struggle:

  • I showed Emma my error checking results
  • She said my error rate was pretty high (and I agreed)
  • She looked at my blib and Laura’s blib with people from her lab (a.k.a. the Skyline creators)
  • They found asterisks at the end of some of the blib sequences that could affect Skyline’s ability to peak peaks
  • Another problem may be that brecan reports the same peptide multiple times, and Skyline doesn’t like that

Overall, this means that my current Skyline output isn’t valid anymore. I can’t work on MSstats or target identification using Skyline data, but I can still look through the literature for proteins of interest. Emma and her team are working on fixing Skyline for our files, so I just have to wait! After everything’s fixed, I will need to check error rates again for the same peptides.

from the responsible grad student http://ift.tt/2pTIeV0
via IFTTT

Yaamini’s Notebook: Protein Extractions Round 2, Part 2

More cowbell? More samples.

I started thinking about my next round of protein extractions a few weeks ago, but now I have a better plan. I’m going to focus solely on the first outplant. I also noticed that the second ouptlant Case Inlet organisms had the lowest sample size. There were only five in the bare treatment, and one in the eelgrass treatment. For this reason, using only the first outplant would give me a larger sample size and allow me to understand the differences between all sites and eelgrass conditions.

I’m going to extract proteins in two blocks of 25 samples each:

Table 1. Proteins to extract. The bolded samples are those I will extract in the first block.

Site Condition 1 2 3 4 5
PG B O25 O83 O54 O51 O52
PG E O31 O71 O78 O56 O30
FB B O36 O70 O43 O40 O35
FB E O64 O46 O32 O24 O49
WB B O129 O126 O135 O121 O122
WB E O140 O145 O147 O131 O144
SK B O111 O120 O99 O96 O113
SK E O103 O101 O106 O102 O91
CI B O11 O13 O16 O21 O22
CI E O01 O08 O10 O06 O04

from the responsible grad student http://ift.tt/2qzxRnV
via IFTTT

Yaamini’s Notebook: Protein Extractions Round 2, Part 2

More cowbell? More samples.

I started thinking about my next round of protein extractions a few weeks ago, but now I have a better plan. I’m going to focus solely on the first outplant. I also noticed that the second ouptlant Case Inlet organisms had the lowest sample size. There were only five in the bare treatment, and one in the eelgrass treatment. For this reason, using only the first outplant would give me a larger sample size and allow me to understand the differences between all sites and eelgrass conditions.

I’m going to extract proteins in two blocks of 25 samples each:

Table 1. Proteins to extract. The bolded samples are those I will extract in the first block.

Site Condition 1 2 3 4 5
PG B O25 O83 O54 O51 O52
PG E O31 O71 O78 O56 O30
FB B O36 O70 O43 O40 O35
FB E O64 O46 O32 O24 O49
WB B O129 O126 O135 O121 O122
WB E O140 O145 O147 O131 O144
SK B O111 O120 O99 O96 O113
SK E O103 O101 O106 O102 O91
CI B O11 O13 O16 O21 O22
CI E O01 O08 O10 O06 O04

from the responsible grad student http://ift.tt/2rlSZzu
via IFTTT