11/19/15

DNA extraction of common garden broodstock:

  1. SS4_2b (redo of extraction attempted on 11/18/15)
  2. NF5_1
  3. NF5_2
  4. NF5_3
  5. NF5_4
  6. NF5_5
  7. NF5_6
  8. NF5_7
  9. NF5_8
  10. NF5_9
  11. NF5_10
  12. NF5_11
  13. NF5_12
  14. NF5_13
  15. NF5_14
  16. NF5_15
  17. NF5_16
  18. NF5_17
  19. NF5_18
  20. NF5_19

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.

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11/18/15

DNA extraction of common garden broodstock. Eluted in 200 uL.

  1. NF3_17
  2. NF3_18
  3. NF3_19
  4. NF3_20
  5. NF4_1
  6. NF4_2
  7. NF4_3
  8. NF4_4
  9. NF4_5
  10. NF4_6
  11. NF4_7
  12. NF4_8
  13. NF4_9
  14. NF4_10
  15. NF4_11
  16. NF4_12
  17. NF4_13
  18. NF4_14
  19. NF4_15
  20. NF4_16
  21. NF4_17
  22. NF4_18
  23. NF4_19
  24. NF5_1
Did the ligation step for Plate 1 of the 2b-RAD broodstock libraries.
1st made fresh annealed adaptors.
Adaptor 1
96x 
5ILL-NR
5
Anti-ILL
5
NFW
490
Adaptor 2
11x
3ILL -NR
5 uL
Anti-ILL
5 uL
NFW
490 uL
Removed 5 uL of digestion product from each well to give 10 uL.
1x
96x
2 uM Adaptor 1
5 uL
480
1 uM Adaptor 2
5 uL
480
T4 ligase
1 uL
96
T4 ligase buffer with 10 mM ATP
4 uL
384
NFW
24
2304
10 mM ATP
1
96
Total
40
3840

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.

  1. SS4_1
  2. SS4_2 (messed up)
  3. SS4_3
  4. SS4_4
  5. SS4_5
  6. SS4_6
  7. SS4_7
  8. SS4_8
  9. SS4_9
  10. SS4_10
  11. SS4_11
  12. SS4_12
  13. SS4_13
  14. SS4_14
  15. SS4_15
  16. SS4_16
  17. SS4_17
  18. SS4_18

When vortexing SS4_2 the cap wasn’t on all the way so most of it splashed out. Need to re-extract.

 

11/16/15 and 11/17/15

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.

  1. NF1_9
  2. NF1_10
  3. NF1_11
  4. NF1_12
  5. NF1_13
  6. NF1_14
  7. NF1_15
  8. NF1_16
  9. NF1_17
  10. NF1_18
  11. NF1_19
  12. NF1_20
  13. NF1_21
  14. NF3_6
  15. NF3_7
  16. NF3_8
  17. NF3_9
  18. NF3_10
  19. NF3_11
  20. NF3_12
  21. NF3_13
  22. NF3_14
  23. NF3_15
  24. NF3_16

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.

1x
96x
10x buffer R
1.2 uL -> 1.5
144
150 uM SAM
.8 -> 1
96
AlfI (2 U/uL)
.5 -> .625
60
water
1.5 -> 1.875
180
Total uL
5

Let digest for 2 hours at 37degC followed by 15 minutes at 65degC then put in the fridge.

Friday 11/13/15

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.

 

Friday 11/6/15

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.

Time
9:00am
-control tank: 9degC
-chilled tank: 0degC; turned off chiller
10:30am
– chilled tank: 3degC
12:00pm
-chilled tank: 5degC
1:00pm
-chilled tank: 7degC
-control tank: 10degC; switched to flowthrough system
2:00pm
-chilled tank: 9degC; added overnight oysters to control tank
-control tank: 11degC

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:

  1. SSB10
  2. NFA7_1
  3. NFA7_2
  4. SSB11
  5. NFA13R: 1.3cm x 1.4cm
  6. HCA10_1: 1.3 x 1.2
  7. HCA10_2: .9 x .7
  8. HCA15 (top)
  9. HCA6 (top)
  10. NFB12 (middle)
  11. NFB13R (2nd from top)
  12. NFB7 (left)
  13. HCA8

Wednesday 11/4/15 and Thursday 11/5/15

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

Chilled treatment on the left and controls on the right

It's cold!

Tiles added to treatments. The numbers represent oysters on that tile after sampling for gene expression. F = front of tile, S = side, B= back
Control
20 hours 0degC
3 hours 0degC
HCA5: 12F, 1S, 13B
HCA13: 9F, 1S
HCA11: 10F, 3S
HCB2: 7F, 2S, 8B
HCA12: 8F, 1S
SSA9: 4F, 1S (4 sampled)
HCA2: 1F, 8B
HCB5: 1
NFA6: 2F (4 sampled)
SSA4: 4F, 2S
HCB8: 2B
SSA13: 4F, 1R
SSA1: 14F, 2S, 1B
SSB11: 1F
SSA11: 0 (4 sampled)
SSA14: 4F, 1S
NFA4: 14F, 1B
SSB2: 0F, 2B
NFA11: 6F, 1B
NFA7: 3F, 1S
NFA9: 4F
NFA12: 3F, 1B
NFB2: 10F
NFA15: 11F, 1B
NFA14: 5F
NFA2: 26F, 1B
NFA?: 15F, 1B
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

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.

Time
10:00AM
– Chiller turned on and set to 0degC.
– Ambient at 11degC
11:30AM
– Chilled side: 7degC
– 350 cultch set oysters placed in chilled water
12:15PM
– Chilled side: 4degC
1:00PM
– Chilled side: 0degC
1:30PM
-Added tiles to control and chilled tanks
4:00PM
-Sorted out ~200 per population; added to new silo in the control tank
– Dissected 10 samples per pop for gene expression

Tuesday 11/3/15: Back in the Field!

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.

HCA11_10_14

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

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.

  • Fidalgo Bay (NF): 215 oysters/50 mL in 450 mL total = 1935 total
  • Oyster Bay (SS): 254/50mL in 250mL total = 1270
  • Hood Canal (HC): 208/50 mL in 325mL total = 1352

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)

Friday 10/30/15

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.

  1. HC5_5
  2. HC5_6
  3. HC5_7
  4. HC5_8
  5. HC5_9
  6. HC5_10
  7. HC5_11
  8. HC5_12
  9. HC5_13
  10. HC5_14
  11. HC5_15
  12. HC5_16
  13. SS3_18
  14. SS3_19
  15. SS3_20
  16. HC1_1
  17. HC1_2
  18. HC1_4
  19. HC1_5
  20. SS3_6
  21. SS2_3
  22. SS2_4
  23. SS2_5
  24. SS3_5

Ran out of tissue for SS2_5, SS3_18, SS3_19, SS2_3, SS3_20.

Quibit samples for concentration and updated sample sheet.

10/28/15

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.

Gel of DNA extraction done on 10/21/15.

  1. S2_9A: 4
  2. S2_10A: 4
  3. S2_11A: 3
  4. S2_12A: 5
  5. S2_13A: 5
  6. S2_14A: 5
  7. S2_15A: 4
  8. S2_16A: 4.5
  9. S2_17A: 5
  10. S2_18A: 5
  11. S2_19A: 5
  12. S3_3A: 5
  13. S3_4A: 4
  14. S3_7A: 4
  15. S3_8A: 4
  16. S3_9A: 5
  17. S3_10A: 4
  18. S3_11A: 4
  19. S3_12A: 2 (low)
  20. S3_13A: 3.5
  21. S3_14A: 4
  22. S3_15A: 5
  23. S3_16A: 5
  24. S3_17A: 4
DNA extraction done 10/23/15

DNA extraction done 10/23/15

  1. NF2_1: 4
  2. NF2_2: 4
  3. NF2_3: 4
  4. NF2_4: 4
  5. NF2_5: 3.5
  6. NF2_6: 5
  7. NF2_7: 4.5
  8. NF2_9: 4
  9. NF2_10: 4
  10. NF2_11: 3.5
  11. NF2_12: 4
  12. NF2_13: 4
  13. NF2_14: 5
  14. NF2_15: 5
  15. NF2_16: 4
  16. NF2_17: 4
  17. NF2_18: 5
  18. NF2_19: 4
  19. NF2_20: 3
  20. NF3_1: 3.5
  21. NF3_2: 3.5
  22. NF3_3: 4.5
  23. NF3_4: 3.5
  24. NF3_5: 3

Did another set of 24 extractions using the EZNA Mollusc kit.

  1. HC4_1
  2. HC4_2
  3. HC4_3
  4. HC4_4
  5. HC4_5
  6. HC4_6
  7. HC4_7
  8. HC4_8
  9. HC4_9
  10. HC4_10
  11. HC4_11
  12. HC4_12
  13. HC4_13
  14. HC4_14
  15. HC4_15
  16. HC4_16
  17. HC4_17
  18. HC4_18
  19. HC4_19
  20. HC4_20
  21. HC5_1
  22. HC5_2
  23. HC5_3
  24. HC5_4