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.

 

Thursday 10/1/15

Took gel slices out of 4degC and they did not look dissolved. Pipetted out the supernatant from each and quantified with HS Qubit. Qubit said the DNA concentration was too low to be recorded, so I added the supernatant back to the respective tubes with gel and did a gel extraction with the Qiagen kit. Added 10 uL of 3M sodium acetate to each sample to bring the pH down. Eluted in 40 uL of provided elution buffer after letting buffer sit in column for 4 minutes. Quantified with HS Qubit:
  • SS2_3: 0.21
  • HC1_4: 0.283
  • HC1_3: .69
  • SS2_4: .281
  • SS2_5: .983
  • HC1_1 : .271
  • HC1_2: .45
  • NF1_1 : .372
In order to multiplex these at equal concentrations, I multiplied the lowest concentration by 38 (the volume after Qubit). I then divided this number by each of the other concentrations to get the volume to add to the pool. Calculations are shown on the 2nd sheet of the Common Garden Samples master list. To concentrate the pooled library, I used a single column from Qiagen Quiaquick PCR Kit. This column was eluted in 30 uL after 5 minute incubation, and was quantified via Qubit at 4.19 ng/uL.

Thursday 8/6/15 and Friday 8/7/15

Thursday 8/6/15

Finally had time to collect the new larvae that I’ve been putting back in the buckets. Most were dead, but can still be used to inform how many have been produced over the past few days. Dumped out all of them. Otherwise caught up on counting from Wednesday and cleaning the tanks that were emptied out.

Friday 8/7/15

Started actively randomizing the order of the tanks on the line. While they were always mixed up when cleaning, now I made sure no tank was in the same place 2 cleanings in a row.

Larval tanks

  • NF_Tank2_160 (224) -> 13,300 total: 500 for DNA, rest to NF_SetB
  • NF_Tank2_160 (100) -> spilled ~400 mL on table. Rinsed what larvae I could off of table onto screen. Swimmers added back.
  • HC_Tank2_160 (224) -> 9,000 total: 500 for DNA, rest added to HC_SetB
  • HC_Tank2_160 (100) -> swimmers only
  • SS_Tank2_160 (224) -> 26,425 total, 425 to DNA, 13,000 to SS_SetB, 13,000 to new silo with cultch
  • SS_Tank2_160 (100) -> swimmers only

Tile Set

Total added

  • SS_SetB: 60,500
  • HC_SetB: 54,925
  • NF_SetB; 34,637

Did counts for NF_SetA, HC_SetA, and SS_SetA.

Wednesday 8/5/15

Had meetings today with Brent, Steven, and Ryan to game plan for the rest of my field season (only 23 more days!). Highlights:

  • Wind down animal husbandry
    • Clean out the “New” tanks
    • Stop collecting new larvae
    • Clean out “160” tanks ASAP
    • Transition cultch set care to hatchery staff
  • Take tissue samples from all broodstock for DNA extractions
    • Store half of samples
    • Try to do as many extractions as possible
  • Make a test 2b-RAD library to run on a MiSeq
    • Order reagents for 2b-RAD libraries, decided on 1/4 reduction
  • Prepare tiles to be placed out in Manchester
    • Once they are all set, randomize among tanks
    • Cull to 20-30 per tile to avoid density impacts on growth
    • Zip-tie tiles to trays with cover
  • Data collection from salinity experiment
    • Talk to Ryann about going through salinity samples for a 2nd live/dead count and to take pictures for size measurement

Very productive chat, but made the rest of the day kind of rushed. Did not get around to cleaning the broodstock or collecting new larvae (although I got rid of the “New” tanks so had nowhere to put them). Decided to max out the SetBs after reaching 60,000 or after 1 week.

Larval Tanks

  • NF_Tank2_160 (224) -> 7,931 total: 500 for DNA, rest to NF_SetB
  • NF_Tank2_160 (100) -> swimmers only added back
  • NF_Tank1_new (100) -> DNA taken, rest dumped out
  • NF_Tank1_new (160) -> NF_Tank2_160
  • HC_Tank2_160 (224) -> 14,525 total: 500 for DNA, rest added to HC_SetB
  • HC_Tank2_160 (100) -> swimmers only
  • HC_Tank1_new (100) -> dumped
  • HC_Tank1_new (160) -> HC_Tank2_160
  • SS_Tank2_160 (224) -> 27,300 total, 500 to DNA, rest added to SS_SetB
  • SS_Tank2_160 (100) -> swimmers only
  • SS_Tank1_new (100) -> dumped
  • SS_Tank1_new (160) -> SS_Tank2_160

Tile Sets

  • SS: 47,500
  • HC: 45,925
  • NF: 21,337

Cultch Set

  • Alice showed me how to screen out larger oysters growing on cultch. Very similiar to screening out larvae tanks, except instead of emptying a tank over a screen you dump the silo out over a screen into a filled sink. The oysters that hold on the screen go into a new silo while those that go through are rinsed onto a smaller screen and then put back in the original silo. Sizes go from 450, 1000, 1600…

Friday 6/26/15 and Sunday 6/28/15

Friday 6-26-15

Larvae in catch buckets:

  • Some: HC3, HC2(maybe), HC4, NF5, HC1, SS5, SS1 (maybe)
  • Lots: NF1, NF2 (bottom), SS2, SS4
  • Added to NF_Tank1, SS_Tank2, and HC_Tank2
  • Combined all the families from a group together again for screening and counting.
  • I also screened out the buckets that the broodstock are kept in while I’m cleaning. All of them had a few thousand larvae in them, and these were added to the appropriate tank.

Larval tank counts:

  • SS_Tank1: over a 140 screen and a 100 screen. Needed to use 2 beakers for the 140 as there were so many. Most of the larvae that were on the 100 screen were dead, so I did not add these back to the tank as they may make the rest of the larvae sick.
  • HC_Tank1: screened them over a 160 micron screen and a 100 micron screen, as this tank has the oldest larvae. I was excited to see that some of them had indeed grown up, as about half of the larvae held on the 160 screen.
  • NF_Tank1
  • While taking the 1 mL samples from the SS_Tank1 beakers, I noticed the pipet tip did not fill up to the 1 mL mark. This is a PSRF pipet, and an older model. I compared the pipet to another one from the molecular lab I knew to be calibrated, and found that the PSRF pipet consistently gave .8 mL instead of 1 mL. This was the same pipet I used on Thursday and the same one Steven used on Wednesday. I’ll ask him if he noticed the pipet tip looking low, but for now I’ve changed the spreadsheet from those 2 days to reflect a drop size of .8.

Larval count data

Cleaning: had Natalie help clean some of the broodstock buckets.

I did not enter the data from my waterproof notebook into my spreadsheet until Sunday. This is when I noticed I had gained ~5,000 larvae in NF_Tank1. I

Sunday 6-28-15

I came in Sunday planning to shift some of the M-W-F cleaning to Sat/Sun-Tues-Thurs so that I have more time for taking samples and setting up the next stage. Unfortunately, the hot water went down again for a few hours which delayed cleaning. I wasn’t able to filter out the newly spawned larvae, but I did filter out all the 100 L larval tanks and counted them.

Observed larvae:

  • Some: NF1, HC3
  • Lots: SS1, HC5, SS2, SS3, HC4, SS4

Thursday 6-25-15

Went to the hatchery in the morning to check for larvae, clean/replace banjo filters, rinse drippers, and replenish algae. As there was a hatchery-wide mortality event after the temperature drop over the weekend, I started 2 new 100 L larval tanks: SS_Tank2 and HC_Tank2. This is because it’s pretty hard to separate out live from dead larvae until the live larvae have grown up a bit. In case the dead larvae still in SS_Tank1 and HC_Tank1 cause additional mortality, I want to start a 2nd replicate where none of the larvae experienced the temperature drop.

  • Checking for larvae in larval catch buckets
    • Maybe/Some larvae: SS1
    • Lots/Noticeable larvae: HC3, HC4, HC1, SS4
    • Also screened NF5 but did not get any larvae
    • Added to HC_Tank2 and SS_Tank2
  • Counting larvae
    • need to get better at estimating ~100 per mL. Some of these counts had over 400 per mL which makes it harder to count and more stressful for the larvae when they’re in the tripour beaker

Larval counts data DNA samples

  • As per my conversation with S. Roberts yesterday, I’m keeping all of the samples from my larvae counts, including those I don’t think are necessary for future genotyping. I’m trying 2 different preservation methods, and will do test DNA extractions next week to look at quantity and quality.
    • 75%/90% ethanol: this was suggested by Jake Heare. I pipet up one of the 1 mL larvae count samples and put it in a 1.5 mL tube. Since I used ethanol when counting, all of the larvae sink quickly to the bottom and I pipet out as much seawater as I can. I continue adding the other larvae count samples and decanting off the seawater in the same fashion. If needed I spin them down briefly with a centrifuge. Then I add 75% ethanol to the tube. A day or 2 after I take out the 75% ethanol and add 90% ethanol. These are stored in -20C.
    • RNALater: As described above, I take out as much seawater as I can and then add about .75 mL of RNALater to the tubes. These are stored in a -80C freezer.

In the afternoon, I stopped by the Roberts lab on the UWashington campus to find some bench space for the test DNA extractions and pick up a HOBO temperature logger to stick in one of my broodstock buckets so I can get a better idea of what they’re experiencing in case a temperature malfunction happens again.