Tuesday, September 2, 2014

Today I attempted to isolate DNA. I had six samples, each labeled 1 through 6:
1 - 5000 larvae
2 - 1000 larvae
3 - 1000 larvae
4 - 1000 larvae
5 - 1000 larvae
6 - 1000 larvae
I removed all the remaining ethanol from each of the six blue tubes. Then, one by one, I added 500 uL of DNAzol and then used a pestle to crush the oyster larvae. I crushed them until they no longer felt gritty. Then, I added an additional 500 uL of DNAzol, so that each of the 6 samples had a total of 1000 uL of DNAzol.

Then, I put all six of the samples in the centrifuge for 10 minutes at 10,000 g. I then transferred the supernatant from each sample into new separate tubes, which I labeled 1 through 6, keeping each sample with the same label after the transfers were made. I kept the tubes that still had the mashed up oyster larvae.

Then, using the tubes with the supernatant, I added 500 uL of 100 % ethanol into each sample tube and mixed it with the DNAzol supernatant by inverting the tubes 8 times each. At this point, it was stated in the protocol that a cloudy precipitate should have been visible, but there was none in any of the samples. Then, I put them all back in the centrifuge for 5 minutes at 5000 g. After this step, a pellet of DNA should have been visible in all of the sample tubes, but there were none in any of them.

The next step was DNA wash. I removed all the 100% ethanol from each tube and then put in 850 uL of 75% ethanol into the tube. I inverted the tube three times and then let it sit for a little bit (however, I forgot to let samples 1 and 2 sit) so that any DNA that is present can settle to the bottom. I then removed the ethanol. I repeated this step so that each DNA sample was washed twice with the 75% ethanol.

Since there were no DNA pellets visible, I kept the supernatant from each sample and transferred them into new separate tubes, which I labeled 1s, 2s, etc. And each sample (numbered 1 through 6) supernatant were transferred into their respective new tubes.

Then, back to the centrifuge for the samples that were washed with the 75% ethanol for 1 minute at 5000 g to get all the ethanol to accumulate at the bottom for better complete removal. After the centrifuge, I used a pipette to remove all the remaining ethanol.

The next step was the DNA solubilization in which I added 25 uL of Nanopure water to each sample tube and let them all sit for 5 - 10 minutes. Then, I used the Nanodrop to see if there were any DNA in any of the samples, which it didn't look too good:
20140902 - gigas larvae gDNA ODs.jpg
The second sample "6" was an accident in activating the Nanodrop after cleaning the surfaces.

So, since the results were not very satisfactory, Sam said that he will be working with the remaining samples that I left. I re-homogenized with DNAzol and pestles the 6 original larvae samples that I left behind in their tubes and then put them on a rotating holder overnight so that they will be constantly agitated. The supernatant samples are still saved, so Sam might also do something with those.

Friday, August 28, 2014

Today I counted out 4 more sets of `1000 larvae. These will all be used in DNA isolation.

Wednesday, August 27, 2014

Today I counted out 5 more sets of 1000 larvae.

Tuesday, August 26, 2014

Today I counted out 4 more sets of 1000 larvae.

Monday, August 25, 2014

Today I counted out another set of 1000 larvae. I also helped Brent by putting Wild Oly Oyster shells into labeled bags.

Thursday, August 21, 2014

Today I counted out two more sets of 1000 oyster larvae and placed them into the blue tubes.

Wednesday, August 20, 2014

Today I put all the rest of the tide data into the excel spreadsheet for Fidalgo. I also finally figured out how to de-clutter the x-axis for the tidal graphs. Here is the graph for Oyster Bay's Tidal Fluctuations over the course of August 17, 2013, through July 31, 2014:
DailyTidesatOYSTERgood.png
(Link to data for graph above: AlltidesatOyster.xlsx 72KB Aug 20 2014 11:56:03 AM)

Tides for Fidalgo:
DailyTidesatFIDALGOgood.png
(Link to data for graph above: AllTidesForFidalgo.xlsx 75KB Aug 20 2014 12:13:59 PM)

For Manchester:
DailyTidesatMANCHESTERgood.png
(Link to data for graph above: ALLTidesManchester.xlsx 77KB Aug 20 2014 12:29:22 PM)


Monday, August 18, 2014

Today I updated some of the graphs that I've been working on for Jake's data and also worked on trying to fix the tide chart graphs. I put the tide data for Manchester into an excel spreadsheet:
ALLTidesManchester.xlsx 62KB Aug 18 2014 11:48:30 AM

And I also started putting in the tide data for Fidalgo:
AllTidesForFidalgo.xlsx 54KB Aug 18 2014 12:10:29 PM

I still haven't been able to successfully de-clutter the x-axis for the graphs… so I will continue to work on that.


Friday, August 15, 2014

Today I learned from Sam how to use the Nanodrop spectrophotometer machine. We used it on the larvae counts that Etilet and I helped count out. There were three tubes filled with 10 larvae each, three with 500 each, and we only got around to one with 1000 larvae. The following links are the data and the graphs from the Nanodrop:
http://eagle.fish.washington.edu/Arabidopsis/20140815%20-%20gigas%20larvae%20gDNA%20plots.JPG <-- Graphs
http://eagle.fish.washington.edu/Arabidopsis/20140815%20-%20gigas%20larvae%20gDNA%20ODs.JPG <-- Data

I also watched Sam quantize… quantitate… DNA from the same larvae samples. The results that we looked at from the machine weren't very satisfactory, so he will be doing it again later this week, and I will observe again.


Thursday, August 14, 2014
Today I watched Sam perform PCR on a sample of bacteriophage that infect abalones infected with withering syndrome that Sam is working on for Friedman's lab.

Wednesday, August 13, 2014

Today I worked with Etilet and separated the oyster larvae into three samples each of 10, 500, and 1000 oyster larvae. We counted them out under the microscope and transferred them into small tubes (name….?). We then removed the ethanol with a pipette and then added 500uL of DNAzol and used a pestle to smash the larvae. After 5 or 6 grindings of the larvae, we added an additional 500uL to the tube, so that each had a total of 1000uL of DNAzol with the mashed up oyster larvae. The DNAzol

Monday, August 11, 2014

Today I finished the tide graph for Oyster Bay and used the same date range as that for the temperature data at Manchester. AllTidesatTracyton.png
It's kind of a mess and I have to work on the x-axis labels because they're just all blobbed together. But the general trends of the tidal fluctuations can be seen fairly easily over time. (Link to Excel File: AlltidesatOyster.xlsx 75KB Aug 11 2014 11:31:13 AM)

Thursday, August 7, 2014

Today I just worked more on graphing the temperature data and tide data. I was able to graph the temperature data from August 2013 to June 2014.
(Link to Excel file: ManAug13toJun14.xls 1734KB Aug 07 2014 01:48:49 PM)
ManchesterTemperatureChart.png
I also worked on entering tidal data into an excel spreadsheet for Oyster Bay. I have been getting the data from this website :
http://www.tides4fishing.com/us/washington/tracyton , and have four more months' worth of data to enter for this site. I am using the same time frame as that of the temperature data, so August 2013 to June 2014. Here's a link to the current progress: Alltidesattempt.xlsx 39KB Aug 05 2014 05:24:36 PM)

Tuesday, August 5, 2014
Today I worked on counting larvae for a few hours. I had an approximately 45mL sample of ethanol with the oyster larvae. I then stirred them in a beaker in one direction and then did a final stir in the opposite direction and immediately used a pipette to take a sample to place in a well for counting under the microscope. I tried doing both 500uL and 1000uL samples to count larvae. The numbers and averages are in this link to the spreadsheet -
https://docs.google.com/a/uw.edu/spreadsheets/d/1cCSCxO-Sz4GhS93cTMfPAVOhV5gQR5lCX7vJPOgtpGE/edit#gid=0

I also worked on making a more detailed temperature and tidal changes graphs. Excel still can't really do much with the temperature because there are too many data entries, so I'll have to ask what to do about that. I worked on making a more detailed tide chart and the rough draft of the tides at Tracyton (Oyster Bay) follows: (Link to excel spreadsheet: Alltidesattempt.xlsx 39KB Aug 05 2014 05:24:36 PM)

DailyTidesApril30toMay30.png
This is just for April 30, 2014 through May 30, 2014. I'll have to figure out how to make it less cluttered and maybe just show one Low and one High per day? I'm not sure yet.

I also looked at the agar plates that Sam and I tried to grow the V. tubiashii on. The samples that were kept in the freezer grew perfectly fine, while the sample that had been used at room-temperature for a while did not have any growth.


Friday, August 1, 2014

Today I did more lab stuff! I played around with this acrylic wheel that is designed to split a volume into two theoretically equal parts, and thus, theoretically, also whatever is suspended in the volume of liquid. In today's case we were trying to split a volume of ethanol with oyster larvae into small enough equal portions for an easier counting experience under a microscope. It was challenging to use the device because the larvae kept getting stuck to the surface of the wheel and it was hard to figure out how to keep them from doing that.

After playing with that for a while, I then tried another method of taking smaller samples of the ethanol and larvae in order to count them under the microscope. This worked fairly well. I used one half of the ethanol and larvae sample. I had sort of succeeded in splitting the original 200mL of ethanol and oyster larvae into one 112mL sample and one ~100mL sample. There was an increase in ethanol because I stopped midway through the process to spray down the inside of the wheel with ethanol in the hopes that it would keep more of the larvae in the sample. I took the sample that contained 100mL of ethanol and stirred it in one direction until it looked fairly suspended, and then once in the opposite direction to try to counteract the tendency of the larvae to accumulate in the middle of the ethanol. I then used a pipette to place 500microliters into a well. I did this process two more times so that I ended with three wells with about 50microliters of ethanol and however many larvae were suspended.

I then counted the larvae in each well. The counts follow:
Well # in Column 2
Count 1
Count 2
Count 3
Average
Well # 1
13
13
13
13
Well # 2
14
14
14
14
Well # 3
22
22
22
22
So this method seems to be a fairly decent option. It was very easy to count the larvae.


Thursday, July 31, 2014

Today I did some lab work! I helped Sam with spreading Vibrio tubiashii across agar plates to try to get some cultures growing. We used both frozen samples and room-temperature samples. I then worked on making some more graphs in excel, but this time just focusing on more detailed temperature data over the course of the sampling dates for Jake's research. Excel is having a rough time dealing with all the data entries, so a graph hasn't been made yet that can be easily read. I will continue to work on this in the next week.

Link to the data:
ManAug13toJun14-2.csv 579KB Jul 31 2014 04:05:06 PM

Wednesday, July 30, 2014

Today I updated the percent brooders with averaged weekly temperature graphs with the new data from Jake's field work last week. I then put the graphs of percent brooders with the temperature and also with the tides into a google doc presentation just for an easier viewing experience.
Link to google doc presentation:
https://docs.google.com/a/uw.edu/presentation/d/1qrGKryRLCdWkpo1ZnP5FVGPqXPVEfkuvk8nTwtYART8/edit#slide=id.g36bcdc8e4_045

Tuesday, July 29, 2014

Today I worked on graphing the relationship between the Percent Brooders separated by site with the tidal cycles. I found the tide information from the following website:

For Fidalgo -
http://www.tides4fishing.com/us/washington/anacortes-guemes-channel
For Manchester-
http://www.tides4fishing.com/us/washington/clam-bay
For Oyster Bay-
http://www.tides4fishing.com/us/washington/tracyton

I took the averaged lowest low tides per sample week dates per site. Then I graphed those against the percent brooder data which are separated through color-coding into the different populations.

The link to the Excel File:
NewPercBrooderswithTidesatSepSites.xlsx 57KB Jul 29 2014 04:14:22 PM

The graphs follow:
PercBrooderswithLowestLowTidesFIDALGO.png
PercBrooderswithLowestLowTidesMANCHESTER.png
PercBrooderswithLowestLowTidesOYSTER.png
It looks like at Oyster Bay that there could be a correlation between the spring tides and the increased brooding in the Southern Population.


Friday, July 25, 2014
Today I figured out how to make the Percent Brooders over Time with Temperature at the different sites separated by population graphs look the way I was trying to make them. I was organizing the data in excel all wrong. So here are the new versions of the graphs:

(Link to Excel file: NewerPercBrooderswithTatSepSites.xlsx 59KB Jul 25 2014 11:39:28 AM)
NewPercBroodwithTatFidalgo.png
NewPercBroodwithTatManchester.png
NewPercBroodwithTatOyster.png
Based on these graphs it seems as though the S Population broods better in warmer temperatures.

Thursday, July 24, 2014

Today I have been playing around with the Percent Brooder data and am having a hard time figuring out how to make the graphs that I want on Excel. I have been working with the Percent Brooder data per site per population and the average weekly temperature per site data.

First, I made some graphs of the average temperature over time.
(Link to Excel File: AverageGperPopOverTime.xlsx 30KB Jul 08 2014 04:09:45 PM)
AverageWeeklyTPerSite.png
AverageTPerWeekAtFidalgo.pngAverageTPerWeekatManchester.png
AverageTPerWeekAtOyster.png

Next, I tried to figure out how to combine this temperature data in line-graph form with the column charts I have previously made of the percent brooders over time. I tried first doing each population separately. So, for each site, I created three separate graphs:
(Link to Excel File for following charts: PercentBroodersSepbyPopwithToverlay.xlsx 60KB Jul 24 2014 04:41:41 PM)

Fidalgo:
PercBrooderswithTatFidalgoPopN.png PercBrooderswithTatFidalgoPopH.png
PercBrooderswithTatFidalgoPopS.png
Manchester:
PercBrooderswithTatManchesterPopN.pngPercBrooderswithTatManchesterPopH.png
PercBrooderswithTatManchesterPopS.png
Oyster:
PercBrooderswithTatOysterPopN.pngPercBrooderswithTatOysterPopH.png
PercBrooderswithTatOysterPopS.png

Then, I tried to do it with all populations combined, but separated by sites with the temperature. I don't know how easy these are to read, but I'm still working on them.
(Link to Excel File with Data: attemptatToverlayonPercentBrooders.xlsx 52KB Jul 24 2014 04:35:39 PM )
PercBrooderswithTatFidalgoAllPops.png
PercBrooderswithTatManchesterAllPops.png
PercBrooderswithTatOysterAllPops.png
The three graphs directly above were really hard to figure out how to make because the data has to be separated by date and also by population, which made the temperature hard to graph because in order for excel to be able to make a line graph, I had to put in each Temperature three times per sample week date since each week is separated into three categories. I don't really like how these graphs look, so I will continue to work on them.

Monday, July 21, 2014

Today I didn't do a whole lot of new stuff, but mostly talked with Steven about what I should focus on in the coming weeks. I will be working mostly with the Percent Brooders data from July 11th post and try to figure out better ways to visualize the data and keep updating as new data comes in week to week. I haven't made too many changes yet, but will be doing so in the next few days. I will also be thinking of ways to relate that data visually with the changes in temperature per site over time.

Link to Percent Brooder data: BroodersovertimeSEPARATESITESgood.xlsx 48KB Jul 21 2014 12:04:16 PM


Wednesday July 16, 2014

Today I made some more adjustments to graphs that I have been working on, and have always made some new graphs. The following show number of spawners per site as the total number for that week, rather than an additive visual over time…
UpDownGraphofSpawnersperSiteperweekOYSTER.png
UpDownGraphofSpawnersperWeekFIDALGO.png
UpDownGraphofSpawnersperWeekMANCHESTER.png
Link to data:
UpandDownGraphofNumSpawnersperdaypersite... 42KB Jul 16 2014 10:32:03 AM

Then, I added the temperature fluctuations over the weeks at those individual sites over the above graphs to try to show any correlation between the two factors:

WeeklyNumSpawnerswithTOverlayOYSTER.png
WeeklyNumSpawnersWithTOverlayFIDALGO.png
WeeklyNumSpawnerswithTOverlayMANCHESTER.png
Link to data:
UpandDownGraphofNumSpawnersperdaypersite... 44KB Jul 16 2014 04:02:10 PM

The graph for Oyster Bay with the addition of the Temperature averages for the sample dates, it looks like the temperature and number of brooders could be correlated, but hard to tell just from what I've done whether anything is really significant.

Also today, I got to count oyster larva! So, I got a little break from the computer work. Both fun, just different kinds of fun. The counts are in the table below.
Well #
Count 1
Count 2
Count 3
Average
1
333
337
322
330.67
2
255
247
249
250.33
3
228
224
229
227

Friday July 11, 2014

Today I tried fixing some of the graphs that I made earlier this week. I had to adjust the axes to be the same so that the values on the graphs could be better compared
PercentBroodersofGNewFidalgo.png
PercentBroodersofGNewManchester.png
PercentBroodersofGNewOyster.png
(Link to data:
BroodersovertimeSEPARATESITES.xls 49KB Jul 14 2014 11:58:47 AM


I also tried making a few more graphs showing the relationship between the number of brooders per population across all sitesNPopTotal#SpawnersAcrossSites.pngHPopTotal#SpawnersAcrossSites.png
SPopTotalSpawnersAcrossSites.png
From these graphs, it looks like the Oyster Bay site is doing pretty well.
Link to excel data sheet: SeparatePopulationSpawnersAcrossSites.xlsx 45KB Jul 11 2014 11:15:52 AM



July 9, 2014

Today I worked on trying to figure out a way to combine the previous three visuals into one. My attempt follows: Total#SpawnersAllPopsTogether.png
I feel like there should be a way for me to make all the points connect into a smoothed-out line, but as of yet, I have not been able to figure it out.
(Link to data: TotalNumSpawnersOverTimeAllPopsinOneGrap... 43KB Jul 14 2014 11:31:48 AM)


July 8, 2014
Today I worked on demonstrating the relationship between the percentage of brooders of gaping oysters per population per site over time. %BroodersatOyster.png%BroodersatFidalgo.png%BroodersatManchester.png
These graphs all seem to show that in general, the Southern population is doing a pretty good job at successful brooding compared to the N and H populations. Especially in Oyster Bay. (Link to data:
BroodersovertimeSEPARATESITES.xlsx 46KB Jul 08 2014 04:43:26 PM
I also looked at showing the total number of brooders per population over time. I separated the graphs into the separate sites and just added the spawners up to create an increasing trend as more brooders appeared. I didn't separate the brooders into the separate populations. Instead these graphs just demonstrate a running total of spawners per site. Total#SpawnersatFidalgo.png

Total#SpawnersatManchester.png
Total#SpawnersatOyster.png
The oysters at Oyster Bay seem to be doing pretty well with spawning compared to the other two sites. ( Link to data:
TotalNumberSpawnersSEPARATESITES-3.xlsx 44KB Jul 15 2014 03:52:12 PM