DNA extraction of common garden broodstock:
Afterwards, got the concentration of these extractions with Qubit. Plated of 1ug of DNA for Libraries 2, 3, and 4 (see “Libraries” sheet of the Common Garden Samples datasheet). Put Airpore tape over the plates and left in an incubator at 37degC.
DNA extraction of common garden broodstock. Eluted in 200 uL.
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Adaptor 1
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96x
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5ILL-NR
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5
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|
Anti-ILL
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5
|
|
NFW
|
490
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|
Adaptor 2
|
11x
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|
3ILL -NR
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5 uL
|
|
Anti-ILL
|
5 uL
|
|
NFW
|
490 uL
|
|
1x
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96x
|
|
|
2 uM Adaptor 1
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5 uL
|
480
|
|
1 uM Adaptor 2
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5 uL
|
480
|
|
T4 ligase
|
1 uL
|
96
|
|
T4 ligase buffer with 10 mM ATP
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4 uL
|
384
|
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NFW
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24
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2304
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10 mM ATP
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1
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96
|
|
Total
|
40
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3840
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Let to digest at 16degC for 2 hours followed by 10 minutes at 62degC and then put in freezer.
In the evening did another set of extractions. These are repeats of the ones done on 10/27/15 that turned out poorly.
When vortexing SS4_2 the cap wasn’t on all the way so most of it splashed out. Need to re-extract.
Monday 11/16/15
Took out the plate from the incubator that was setup on Friday 11/13/15. The samples were completely dried down. I added 10 uL NFW to each well and left the plate in the fridge overnight. I also Qubit-ed more DNA extractions.
Tuesday 11/17/15
DNA extraction of broodstock from common garden experiment.
Eluted these in 200 uL to increase yield. Used Qubit to get concentration.
Set up a digestion of the 1st 2bRAD library plate. To save time, I did not take out 2 uL from each well to make 8uL as listed in the protocol. Instead I increased all of the master mix reagents so they would add up to 5uL, therefore maintaining a 2:1 sample:master mix ratio.
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1x
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96x
|
|
|
10x buffer R
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1.2 uL -> 1.5
|
144
|
|
150 uM SAM
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.8 -> 1
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96
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AlfI (2 U/uL)
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.5 -> .625
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60
|
|
water
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1.5 -> 1.875
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180
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|
Total uL
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5
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Let digest for 2 hours at 37degC followed by 15 minutes at 65degC then put in the fridge.
Starting to setup the 2b-rad libraries of adult broodstock that will be sent off in a couple weeks for sequencing. I made a sheet labelled “Libraries” on the Common Garden DNA Samples datasheet outlining which sample will go in which library. With our barcodes we can sequence up to 96 samples per lane. I decided to do about 75 per lane across 4 lanes, mixing up the 3 populations across the lanes. I’ll also repeat 3 samples across all lanes and repeat 2 samples within each lane.
I got the concentration of the extractions for NF2 with Qubit and then pipetted out 1 ug of DNA for the samples in Library 1 in a 96 well plate. I put Airpore tape over the plate and left it in a 37degC incubator to dry out.
At 9:00am I turned off the chiller and took a sample of oysters from the overnight treatment. It appeared that many of the oysters from the overnight treatment were gaping slightly, but they still offered resistance during dissection. I dissected 6 cultch set oysters per population and 4 tile set oysters and put the whole body tissue in RNALater. I also dissected 10 oysters per population from the control treatment. Instead of moving the overnight oysters over to the control tank, I let the temperature gradually increase by draining some of the tank and adding ambient water. We switched the control tank over to a flow-through system with live algae dripping in. At 2:00pm I moved the overnight chilled oysters to the control tank. The hatchery folks are going to keep an eye on the oysters for 2 weeks, after which they’ll sample all of the survivors.
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Time
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9:00am
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-control tank: 9degC
-chilled tank: 0degC; turned off chiller
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10:30am
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– chilled tank: 3degC
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12:00pm
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-chilled tank: 5degC
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1:00pm
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-chilled tank: 7degC
-control tank: 10degC; switched to flowthrough system
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|
2:00pm
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-chilled tank: 9degC; added overnight oysters to control tank
-control tank: 11degC
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I took apart the tile trays and drained out the tank they had been in. Some tiles that had only a few oysters on them were not used in the temperature experiment. I dissected these and put the whole body in RNALater, figuring they could be used for genotyping or looking at epigenetic in the F2 generation. Some of them I took a picture for size data and some of them I measured with a ruler.
Samples from tiles:
Wednesday 11/4/15
Gave a talk to the PSRF monthly meeting in the morning. Afterwards I went with Alice to the dock and brought up the cultch set F2 oysters to the hatchery. As there weren’t very many tile set oysters, I wanted to supplement the experiment with these. I put them in one of the setting tanks outside in static, ambient water.
Thursday 11/5/15 (Experiment Day)
Alice and I built a little “house” for the chiller to protect it from the rain. We set up the chiller in the smaller section of the setting tank in order to cool down the water quicker. The chiller was set to 0degC and turned on at 10am. Water on the other side was at 11degC, a little cooler than ambient due to the air temperature overnight. For the controls, 250+/- 5 cultch set oysters from each population were placed in 180 micron silos hanging in the ambient temperature side of the tank. I put around 350 oysters in silos on the chilled side at 11:30am when the temperature was at 7degC. At 1:00pm the temperature was at 0degC and maintained that temperature all day. At 1:30PM I added some tiles to the control and chilled tanks.
Chilled treatment on the left and controls on the right
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Control
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20 hours 0degC
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3 hours 0degC
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HCA5: 12F, 1S, 13B
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HCA13: 9F, 1S
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HCA11: 10F, 3S
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HCB2: 7F, 2S, 8B
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HCA12: 8F, 1S
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SSA9: 4F, 1S (4 sampled)
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HCA2: 1F, 8B
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HCB5: 1
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NFA6: 2F (4 sampled)
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SSA4: 4F, 2S
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HCB8: 2B
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|
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SSA13: 4F, 1R
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SSA1: 14F, 2S, 1B
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|
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SSB11: 1F
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SSA11: 0 (4 sampled)
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|
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SSA14: 4F, 1S
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NFA4: 14F, 1B
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|
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SSB2: 0F, 2B
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NFA11: 6F, 1B
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|
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NFA7: 3F, 1S
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NFA9: 4F
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|
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NFA12: 3F, 1B
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NFB2: 10F
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|
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NFA15: 11F, 1B
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NFA14: 5F
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|
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NFA2: 26F, 1B
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||
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NFA?: 15F, 1B
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||
|
Total HC: 52
Total SS: 19
Total NF: 63
|
Total HC: 22
Total SS: 17
Total NF: 41
|
Total HC: 13
Total SS: 5
Total NF: 2
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At 4:00pm I sorted out 200 cultch set oysters from the chilled tank and added them to new silos in the control tank labelled with the population name and “3 hr chilled”. In addition to looking for differential mortality, we are interested in looking at temperature-induced gene expression. I put some oysters from each group in small (300 mL) cups with chilled water and then dissected 6 from each population and put whole body tissue in 1.5 mL tubes with RNALater. This took about 45 minutes total but I tried to alternate between populations while dissecting. I took pictures of most dissected oysters with a ruler. At 5:00pm I brought in one tile for each population from the chilled tank and dissected 4 oysters per population, giving 10 samples each. These samples were placed in the fridge overnight. The other ~150 oysters in the chilled tank were left there overnight to be sampled the next day.
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Time
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|
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10:00AM
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– Chiller turned on and set to 0degC.
– Ambient at 11degC
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|
11:30AM
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– Chilled side: 7degC
– 350 cultch set oysters placed in chilled water
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|
12:15PM
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– Chilled side: 4degC
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|
1:00PM
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– Chilled side: 0degC
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|
1:30PM
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-Added tiles to control and chilled tanks
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|
4:00PM
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-Sorted out ~200 per population; added to new silo in the control tank
– Dissected 10 samples per pop for gene expression
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On Sunday 11/1/15 I took an early flight out to Seattle for a week of torturing oysters. My goals for the week were to take pictures for size of my juvenile F2 oysters and do an experiment to test for differences in resilience to cold temperature shock.
Over the summer I set juvenile Olympia oysters on PVC tiles and hung them off the dock by the Manchester Research Station at the end of the summer. Unfortunately, one of my stacks of trays fell off the dock a few days after during a crazy storm. They sat on the bottom for a couple of days and then were rescued by a crew that left them sitting on the dock in the hot afternoon sun. Once the hatchery crew realized where they were they quickly put them in a tank, but the stack may have been out of water for a few hours. They were redeployed on 9/10/15 with safety line and haven’t had an issue since.
On 10/14/15 two of the PSRF hatchery crew, Stuart and Laura, pulled up the trays and photographed the tile to get size information. There was a lot of mortality on the stack that had been out of the water, but the other stack had good survival.
Picture taken on 10/14/15 of a tile with Hood Canal juvenile oysters.
Today I pulled up the trays and took pictures of the tiles. It’s exciting to see how much they had grown, even in just 3 weeks!
Picture of Hood Canal oysters taken 11/3/15
I left the oyster tiles in a large, static tank at ambient temperature and splashed in some live algae. Alice and I also checked on some F2 oysters that were set on cultch at the end of the summer. These were basically leftover larvae after I had enough in my tanks with tiles. As larval production was really ramped down at the end of the summer, I suspect that these are from only a few individuals per population. To get a rough estimate of how many there were per population, I measured out 50 mL of oysters, counted them, and then measured the total volume of oysters.
At 9:30am we turned on a chiller in one of the outdoor setting tanks and set it to 0degC. I monitored it throughout the day to see how long it took to get to 0.
|
Time
|
Degrees C
|
|
9:30AM
|
13
|
|
11:00AM
|
8
|
|
12:00PM
|
6
|
|
1:15PM
|
3
|
|
2:30PM
|
0.5
|
|
3:00PM
|
0 (turned chiller off)
|
Finished up the Hood Canal broodstock extractions and did some re-extractions of ones that I had either used up when making test PCR libraries or had come out degraded. Used the EZNA mollusc kit.
Ran out of tissue for SS2_5, SS3_18, SS3_19, SS2_3, SS3_20.
Quibit samples for concentration and updated sample sheet.
Did another set of 24 extractions using the EZNA Mollusc kit.
Qubit of extractions done on 10/21/15 and 10/28/15. Updated the sample sheet with concentrations.
Ran out gels for DNA extractions that had been done on 10/21/15 and 10/23/15.
Gel of DNA extraction done on 10/21/15.
DNA extraction done 10/23/15
Did another set of 24 extractions using the EZNA Mollusc kit.
Getting My Genes Wet 