Yaamini’s Notebook: SRM Dilution Calculations

My prior calculations were incorrect!

As I mentioned previously, Laura and I need to run dilutions of our samples to confirm that as the concentration of protein in our samples decreases, the assay does not detect them as intensely. When I showed Emma my calculations, she suggested making dilutions based on protein concentrations (µg) as opposed to µL of sample added. Here are my final numbers:

Table 1. Dilution calculations

Vial Dilution Ratio µg to Inject µL to Inject µg oyster Needed µg geoduck Needed µL oyster needed ((µg/µL)x15 µL/1 µg/µL) µL geoduck needed ((µg/µL)x15 µL/2 µg/µL) PRTC added µL ACN Total Volume (µL)
1 10:1 1 1 0.9 0.1 13.5 0.75 1.5 N/A 15.75
2 7.5:1 1 2 0.87 0.13 6.5 0.5 0.75 7.25 15
3 5:1 1 2 0.8 0.2 6 0.75 0.75 7.5 15
4 2.5:1 1 2 0.6 0.4 4.5 1.5 0.75 8.25 15
5 1:2.5 1 2 0.4 0.6 3 2.25 0.75 9 15
6 1:5 1 2 0.2 0.8 1.5 3 0.75 9.75 15
7 1:7.5 1 2 0.13 0.87 1 3.25 0.75 10 15
8 1:10 1 1 0.1 0.9 1.5 6.75 1.5 5.25 15

For PRTC, our stock concentration volume is 0.5 pmol/µL. I changed the injection volume for the 10:1 and 1:10 dilutions from 2 µL to 1 µL to ensure we were pipetting volumes over 0.5 µL. This made it so that the final volume for the 10:1 injection was 15.75 µL, but Emma said that was okay.

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Yaamini’s Notebook: Revised Spawning Plan

Featuring reduced crosses and pools

After showing Steven and Brent my preliminary spawning plan, I used their feedback and Joth’s comments from the July hatchery update meeting to make a revised plan. I was also able to think through the actual steps of spawning and materials needed for this plan because I shadowed a C. gigas spawn at Taylor Shellfish on Monday.

Crosses and pools

Here are the current oyster counts in my conditioning tanks.

Table 1. Oyster counts in each tank.

Tag Label A B Total
1 7 9 16
2 8 8 16
3 6 6 12
4 6 7 13
5 9 8 17
6 5 8 13
Heat Shock 5 6 11

Assuming there is a 50/50 sex ratio, I calculated the number of males and females I’d have from each tank. I assumed I would have more males than females, just for the numbers.

Table 2. Sex ratios for each tank.

Tag Label M F Total
1 8 8 16
2 8 8 16
3 6 6 12
4 7 6 13
5 9 8 17
6 7 6 13
Subtotal 45 43 88
Heat Shock 6 5 11
Total 51 48 99

To make my crosses, I will pool all of the eggs from my six tanks and from the heat shock treatment. Keeping males separate, I will cross all of the males from Tanks 1-6 with the females from Tanks 1-6. Separately, I will cross the heat shock oysters with eachother. Therefore, I will have (45 males)(6 egg pools) + (6 males)(1 egg pool) = 276 crosses.

After measuring hatch rate for each cross, 24 hours post-fertilization, I will pool hatched D-hinge larvae from each cross according to Table 3.

Table 3. Treatment pools for crosses.

Pool Parent 1 Parent 2 Parent 1 Tank Parent 2 Tank
1 Low pH Female Ambient pH Male Tanks 1-3 Tanks 4-6
2 Ambient pH Female Low pH Male Tanks 4-6 Tanks 1-3
3 Low pH Female Low pH Male Tanks 1-3 Tanks 1-3
4 Ambient pH Female Ambient pH Male Tanks 4-6 Tanks 4-6
5 Heat Shock Female Heat Shock Male Heat Shock Tank Heat Shock Tank

Once pooled, I will count the number of live and dead larvae in each pool. Based on these counts, I will calculate how many larvae I need to add to each bucket to get a density of 1/2 larvae per mL. I will then redistribute larvae into the static system.

Static system description

For the static system, I want to keep larvae in five gallon buckets. Each pool listed in Table 3 will have five replicates, meaning I need 25 buckets total. Buckets will be placed in larger totes with water and immersion heaters to keep water temperature at 25 ºC for the larval period. I think five buckets will fit nicely into a tote, but I need to check. I’m planning on keeping the totes on the middle table where Laura had her buckets.

Materials needed

  • Shucking knives: 4
  • Oyster separators: Enough to hold 200 oysters
  • Glass capilary tubes: 150
  • Tripours: ~300
  • Scoring knives: 4
  • Spatulas: 4
  • Microscope: 1
  • 80 micron screens: 2
  • 20 micron screens: 2
  • 5 gallon buckets: 50
  • Totes: 8
  • Immersion heaters: 6

Questions

  • Should I try and separate crosses between conditioning tanks (Tank A and Tank B)? I’ve had more mortalities in Tank A.
  • Should I include Ambient x Heat shock crosses?
  • Are we rearing larvae in a static system for the full duration, or are we going to switch to a flow-through at 100 microns?

Ask Molly and/or Rhonda

  • How to calculate the amount of sperm and egg to use when fertilizing
  • An easy way to calculate hatch rate

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Laura’s Notebook: SRM mass spec refresher, prepping samples with PRTC

Mass Spec refresher, final sample prep (+PRTC, transferring to autosampler vials)

Prepare PRTC from stock solution to add to samples:

  • As in the DIA preparation I did the following with Peptide Retention Time Calibration Mixture ThermoFisher #88320, stock concentration is 0.5pmol/ul.
  • Pipetted 20ul of PRTC into new tube – the PRTC I used was in Emma’s box, and was an aliquot of 20ul.
  • Added 30ul acetonitrile, vortexed & centrifuged lightly
  • Final volume is 50ul of a [200fmol/ul]
  • Pipetted 1.88ul into each autosampler vial
    • Oh NO! I forgot that i needed to first pipette my mixtures into centrifuge tubes, mix, then transfer to the autsampler vial. I salvaged as much diluted PRTC as I could and restarted.
  • I needed more PRTC. There was another aliquot of stock PRTC in my sample box (as per Yaamini). It was not a full 20ul. I pipetted 10 ul stock PRTC into my PRTC dilution tube, then added 15ul “Final Solvent,” vortexed then spun down. Thus, I ended up using a mix of: salvaged initial mix of PRTC + 25ul second preparation.

Prepared PRTC, Sample, and Final Solvent mix

Labeled new mini centrifuge tubes (0.75ul) and autosampler vials with my sample #’s, organized them in the order I will run them. I will add PRTC, sample & final solvent to the centrifuge tubes, mix, then transfer to the autosampler vials.

Equations from Emma to calculate volumes to add:

  • How much PRTC volume should be added?
    • (PRTC final concentration)(final volume)(1/PRTC stock concentration), i.e. (25 fmol/μl)(15 μl)(1/200 fmol/μl) = 1.88ul PRTC for all my geoduck samples
  • How much sample should be added to each autosampler vial?
    • ((μg to be injected/injection volume)final volume)/sample concentration, i.e. ((1 μg/2 μl)15 μl)/concentration
  • How much Final Solvent to add to each autosampler vial? Final volume should be 15ul
    • 15ul – vol PRTC – vol Sample
  • Calculations in “Samples for SRM” spreadsheet
  • Samples G002 & G016 concentrations were low, and thus the vol sample was >15ul. In these cases I added 3.13ul sample (15ul – 1.88ul PRTC).
  • Worked with the first 25 samples on my randomized list- the rest I kept in the freezer and will prep on Wednesday or Friday (pending PRTC delivery). Here’s the vol of each sample, PRTC, and Final Solvent added. Highlighted samples were low in concentration and thus required irregular volumes: SRM Samples

Bubbles

When I set my pipette to 15ul I consistently was getting a bubble, likely b/c I had a hard less than 15ul. So, I set my pipette to 14.8, and this helped. However, in a handful of samples a bubble got into the autosampler vial and I couldn’t pop it. So, I pulled the mixture out of the autosampler vial, returned it to the centrifuge tube, centrifuged it to remove bubbles, then re-pipetted it into the autosampler vial.

Mass Spec Refresher / notes

  • Loaded my samples in the mass spec tray as per the below map. NOTE: Do not open door when mass spec is “injecting”
1 2 3 4 5 6 7 8
A Blank QC
B O01 O12 O22 O113 O118
C O26 O90 G013 G120 G047 G017 G079 G127
D G060 G009 G002 G128 G016 G071-A G114 G045
E G132 G031 G012 G116 G043 G015 G040 G110
F G008 G109 G122
  • Emma prepared my methods file using the transitions I sent her and saved on the mass spec computer
  • Edit the sequence table to assign sample vial location, sample & file name, vol to inject, and method to use. E.g.:
    • File name: auto-fills based on previous line; can edit if necessary. I’m sticking with Emma/Yaamini’s naming convention of “2017July10_bivalves#” where # increases sequentially for samples AND blanks.
    • Comment: sample ID, e.g. G013
    • Path: directory where data will be saved.
    • Instrument method (Inst Meth): path to the method file – Emma prepped this for me
    • Position: Tray:row,column – for example my G013 sample is located in, 1:C,3
    • Injection Volume (Inj Vol): 2ul for samples, 3ul for blank and QC

SRM Sequence Table

  • The above image shows my sequence for the first 25 samples, starting below the highlighted cell. You’ll see a pattern: Run 5 samples, then a QC, then a blank. Repeat.
  • NOTE: if anything in the Acquisition Queue needs to be edited, it’s best to 1) delete existing, incorrect sequence row in the acquisition queue, 2) create a new row in the sequence table with the correct info, 3) add to the acquisition queue.

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Laura’s Notebook: Second SRM sample prep

Readied last 25 peptide samples for mass spec

0) Labeled mini centrifuge tubes with second batch of samples, and reviewed calculated volumes of sample & Final Solvent to add to each: image

1) Borrowed one last 20ul aliquot of PRTC stock from Emma/Jenn – PRO TIP: order mass spec supples way in advance! 2) Added 30ul Final Solvent, vortexted, spun down 3) Pipetted 1.88ul diluted PRTC to centrifuge tubes 4) Pipetted designated volume of Final Solvent, as per my calculates 5) Got samples out of freezer, pipetted designated vol. into centrifuge tubes as per my calculations; vortexed, centrifuged lightly. 6) Transferred mass spec-ready mixtures to autosampler vials; instead of pipetting 15ul I used 14.7, since using 15ul consistently resulted in air bubbles. Despite this, I had to re-pipette samples 54 & 55. 7) Added these newly prepared samples to the sequence table, continuing the pattern of QC, blank, 5 samples, repeat. 8) Loaded the samples on to Tray 2 in the following order:

1 2 3 4 5 6 7 8
A
B G041 G066 G105 G032 G129 G054 G081 G003
C G074 G014 G049 G053 G104 G055 G042 G064
D G073 G057 G007 G070 G001 G071-B G062
E GBlank OBlank
F

9) Added 65ul Final Solvent to the blank vial; there should be ~130ul blank as of 5:30pm today.

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Yaamini’s Notebook: Manchester Conditioning Update 8

Temperature is at 23 ºC!

On Monday, Grace went out to Manchester and increased the setpoint on the left heater to 27 ºC. Olivia vacuumed the oysters and did not find any mortalities. Dana fed the oysters the remaining 350 mL of Reed’s paste. On Tuesday, Ryan noticed that the algae dosing pump lost it’s prime sometime overnight. He fixed it so it was working when I got to the hatchery. Temperature’s been peaking at 23 ºC every day, but it still drops to about 22 ºC due to diurnal fluctuations. When I checked the heater setpoints this morning, I saw they were heating the water a few degrees hotter than my goal.

img_7993

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Figures 1-2. Heater setpoints upon arrival.

I increased the setpoint on the left heater by one degree just to smooth out fluctuations and have temperature hover at 23ºC during the day.

img_7995

Figure 3. Heater setpoint after increasing it.

Because the oysters haven’t had much food over the past few days, I wanted to feed them really well. I filled the algae header tank with 800 mL of Reed’s for one day and set the dosing rate at 60%. I vacuumed the oysters and found 5 dead :0 Two were from Tank 6A, and three were from Full Ambient A.

Table 1. Revised oyster counts in each tank.

Tag Label A B Total
1 7 9 16
2 8 8 16
3 6 6 12
4 6 7 13
5 9 8 17
6 5 8 13
Heat Shock 5 6 11
Full Amb 4 8 12
Spare 2 3 5
Total 52 63 117

My original plan today was to shuck some oysters, look at gonad development and sex them. Since the oysters that died had died recently, I figured I might as well examine their gonads! Out of the five that died, four were still mostly intact. All four had nice opaque white gonads, one of which was quite milky! I think with increased feeding and temperature they’ll become even milkier. I wanted to sex the oysters, but the microscope at PSRF didn’t let me zoom in enough. Plus it was difficult to pull a bit of the gonad from the degrading oysters. I was able to pull gonad from one oyster, and I think it was a male.

img_7987

img_7988

img_7989

img_7990

Figures 4-7. Gonadal development for oyster mortalities.

img_7992

Figure 8. Gonad under the microscope for first oyster pictured above. I couldn’t zoom in enough, but I believe this is a male gonad.

The next time I go out to Manchester, I’ll bring better tools to pull gonad and see if I can get help from Dana to look at the gonad.

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