I had my Fall 2016 committee meeting on Thursday, Dec. 8. Pretty productive, with some good discussion about what to do going forward with my samples from this summer’s field season. Slides can be found here.
A few weeks ago I made 2 more Genotype-by-Sequencing libraries for my Olympia Oyster population structure study, made up mostly of redos and some samples from the sister species Ostrea conchaphila. I tried out the free version of Benchling for it and really liked the interface and how it organizes protocols with your notes. I’ll continue using it for my wet lab notes and post the links to my entries here. All field work posts and analysis updates will still be posted here. The link to my Benchling is:
This link is on my About page as well.
Monday 8/10/15
Larvae tanks
Tile Set
New larvae
Tuesday 8/11/15
Animal husbandry
Lab work
Extracted DNA from 24 larvae samples that would be good candidates for test 2b-RAD libraries. I chose sets of new larvae (“LC” for larvae catch), “160”s, and “224”‘s with 3-5 days in between.
| Population | Tank | Family | Size | Date | Storage | Est. # | Date extracted | |
| 1 | Hood Canal | LC | >100 | 7/13/2015 | 75/95 EtOH | 8/11/2015 | ||
| 2 | Fidalgo Bay | NA | LC | 100 | 7/13/2015 | 75% EtOH | 8/11/2015 | |
| 3 | South Sound | NA | LC | 100 | 7/13/2015 | RNALater | 8/11/2015 | |
| 4 | Hood Canal | NA | HC2 | 100 | 7/17/2015 | RNALater | 8/11/2015 | |
| 5 | Hood Canal | NA | LC | 100 | 7/17/2015 | RNALater | 8/11/2015 | |
| 6 | South Sound | NA | LC | 100 | 7/17/2015 | RNALater | 8/11/2015 | |
| 7 | Hood Canal | NA | LC | 100 | 7/20/2015 | RNALater | 8/11/2015 | |
| 8 | Fidalgo Bay | NA | LC | 100 | 7/20/2015 | RNALater | 8/11/2015 | |
| 9 | South Sound | NA | LC | 100 | 7/20/2015 | RNALater | 8/11/2015 | |
| 10 | Hood Canal | HC_Tank1_160 | NA | 160 | 7/20/2015 | RNALater | 8/11/2015 | |
| 11 | Fidalgo Bay | NF_Tank1_new | NA | 160 | 7/20/2015 | RNALater | 8/11/2015 | |
| 12 | South Sound | SS_Tank1_new | NA | 160 | 7/15/2015 | RNALater | 8/11/2015 | |
| 13 | Hood Canal | HC_Tank1_new | NA | 160 | 7/24/2015 | RNALater | 8/11/2015 | |
| 14 | Fidalgo Bay | NF_Tank1_new | NA | 160 | 7/24/2015 | RNALater | 8/11/2015 | |
| 15 | South Sound | SS_Tank1_new | NA | 160 | 7/24/2015 | RNALater | 8/11/2015 | |
| 16 | Hood Canal | HC_Tank1_new | NA | 160 | 7/27/2015 | RNALater | 8/11/2015 | |
| 17 | Fidalgo Bay | NF_Tank1_new | NA | 160 | 7/27/2015 | RNALater | 8/11/2015 | |
| 18 | South Sound | SS_Tank1_new | NA | 160 | 7/27/2015 | RNALater | 8/11/2015 | |
| 19 | Hood Canal | HC_Tank2_160 | 224 | 8/3/2015 | RNALater | 8/11/2015 | ||
| 20 | Fidalgo Bay | NF_Tank2_160 | 224 | 8/3/2015 | RNALater | 8/11/2015 | ||
| 21 | South Sound | SS_Tank2_160 | 224 | 8/3/2015 | RNALater | 8/11/2015 | ||
| 22 | Hood Canal | HC_Tank2_160 | >224 | 8/7/2015 | RNALater | 350 | 8/11/2015 | |
| 23 | Fidalgo Bay | NF_Tank2_160 | >224 | 8/7/2015 | RNALater | 504 | 8/11/2015 | |
| 24 | Oyster Bay | SS_Tank2_160 | >224 | 8/7/2015 | RNALater | 641 | 8/11/2015 |
I still have to go back over my notes to get estimated number for some of them. Storage was mostly in RNALater. A lot of the samples had some white precipitate on the bottom. If larvae weren’t in the precipitate I sucked it out before adding the lysis buffer/proteinase K. Also had the same issue previously with larvae being buoyant in the RNALater even after a spindown. Halfway through trying to siphon off as much liquid as I could, I did some research and found that addition of some ice-cold PBS will change the density of the liquid and allow the larvae to settle out. This worked really well and was done for 2,10,13,15,19,20,21,24. It also dissolved most of the white precipitate.
Followed the protocol I listed here, with a 2.5 hour digest. Could not findd the gel rig set-up (found out later it’s in a different building).
Setting System
Last week, I decided to make a 2nd tank with tiles for each of the populations. This stemmed from not seeing as many setters as I’d like on the tiles in the 1st set-up, and because there were still so many in the “160” tanks. Also, having 2 tanks per population can mitigate “tank effects”- possible tank-specific differences that might confound population differences. I picked up some more PVC sheet, had it cut into 4″ x 4″ tiles at the store, and roughed them up in between today’s tank cleanings. To ensure enough larvae set per tile, I’m going to add up to 60,000 larvae per tank over the course of 1 week. While this means some larvae will be a few more days older than others, after an extended period of time this will be insignificant for measuring growth rate.
Did counts for NF_SetA and HC_SetA to get an idea of how many were left to set (ran out of time for SS_SetA).
Larvae tanks
Measured out the “160” tanks over 224, 200, and 100 to get an idea of how many 224s I will get over the next week.
Nine larvae/setting tanks to clean! Michael and I had an evening flight out east for his cousin’s wedding, so today was a pretty rushed day trying to get everything done and still make the 4:05p ferry.
Larvae tanks
Cultch Setting System
Set up a 2nd silo replicate for all 3 populations. As SS only had ~11,000 224-sized larvae, I added 11,000 larvae to each of the silos. 5,781 extra HC larvae were added back to HC_Tank2_160. With NF, there were ~9,000 dead 224-sized larvae- most likely due to my adding the the ones I wasn’t using back in to Tank2_160. If a larva that is ready to set can’t find an appropriate surface, it will eventually die. I dumped out the 19,700 larvae that were not used in the cultch set (see previous post for my regret about this).
New larvae
Only saw some larvae in HC and SS. I ran out of time to take samples for counting, so just screened out the larvae, cleaned the broodstock buckets, and dumped the larvae back into the buckets.
On Wednesday night I left to spend a few days in Chicago to start setting up the Committee on Evolutionary Biology’s new high performance computer, so Steven Roberts and Sam White came to the hatchery to do the Friday cleaning and counting. I wrote up some instructions– also good exercise so we can reference them when writing up Materials and Methods. Check out their great write-up here (lots of pictures!).
Placed through Pritzker lab
Crassostrea gigas:
Ostrea
Sequences were too low of quality (b indicates reverse)
Realized I didn’t need to do forward and reverse because sequences are so different between Crassostrea and Ostrea that I do not need to confidently call every single nucleotide.
