Wednesday 10/14/15

DNA Extraction of Oly Broodstock

  • SS1_1
    SS1_2 (not tan)
    SS1_3
    SS1_4
    SS1_5
    SS1_6
    SS1_7
    SS1_8
    SS1_9
    SS1_10
    SS1_11
    SS1_12
    SS1_13
    SS1_14
    SS1_15
    SS1_16
    SS1_17
    SS1_18
    SS1_19
    SS1_20
    SS3_1
    SS3_2
    SS3_5
    SS3_6
  • Found 2 tubes labelled SS1_2. Marked the unextracted one with a tan sticker
  • Could not initially find SS3_3 or SS3_4 but found later and reorganized box
  • Left in 60degC water bath for 3 hours
  • Eluted 50 uL then 100 uL

Primers

  • Received the rest of the 2b-RAD primers and resuspended them to 100 uM.
    • BC11
    • BC12
    • HT3
    • HT4
    • HT5
    • HT6
    • HT7
    • HT8
  • Made new 1 uM aliquots of BC2-10 and HT1
    • 1 uL stock + 99 uL NFW
  • Made new 10 uM  aliquots of Lib 1 and Lib 2
    • 2.5 uL stock + 22.5 uL NFW

PCR for 2b-RAD MiSeq run

Used 10 samples from 9/28/15 digestion and ligation. Numbers in parentheses are so I know which tube from the ligation each sample is in.

  1. SS2_6 (5)
  2. SS2_7 (6)
  3. SS2_8 (7)
  4. HC1_5 (8)
  5. HC1_6 (9)
  6. HC1_7 (10)
  7. HC1_8 (11)
  8. NF1_4 (14)
  9. NF1_5 (15)
  10. NF1_6 (16)

Master Mix

1x
10.5x
NFW
23 uL
241.5
10 mM (each) dNTPS
2
21
10 uM ILL-Lib1
2
21
10 uM ILL-Lib2
2
21
5X Q5 buffer
20
210
Q5 Taq polymerase
1
10.5
Added 50 uL of MM to 40 uL of ligation product. Added 5 uL of 1uM BC and HT to each sample, using BC1-10 and HT1.
Run out gel of 2b-RAD PCR product
This time, set up a medium gel for 8 of the samples and a small gel for the other 2. Made a 2% TAE gel, and after running it at 110V for 1 hour let the gels soak in water with 6 uL of EtBr to increase staining. PCR used 19 cycles.

Gel of 2b-RAD PCR product

Gel of 2b-RAD PCR product

20151015_124156

I think I should make sure to mix in the EtBr when doing the post staining as that is likely why there are bright white sections on the gels. It was still much easier to see the bands though. Cut out the bands and left them in 1.5 mL tubes in 4degC for gel extraction on Friday.

Wednesday 9/23/15

  • Did more extractions of broodstock samples. I think once this test library is done the broodstock libraries should be 1st priority, with samples from the stressor experiment 2nd and other larvae samples 3rd.
    • Used the EZNA kit with a 4 hour digestion time. All the tissue for HC1_16 was used up.
  1. HC1_11
  2. HC1_12
  3. HC1_13
  4. HC1_14
  5. HC1_15
  6. HC1_16
  7. HC1_17
  8. HC1_18
  9. HC1_19
  10. HC2_1
  11. HC2_2
  12. HC2_3
  13. HC2_4
  14. HC2_5
  15. HC2_6
  16. HC2_7
  17. HC2_8
  18. HC2_9
  19. HC2_10
  20. HC2_11
  21. HC2_12
  22. HC2_13
  23. HC2_14
  24. HC2_15
  • Finished the ethanol precipitation started on Tuesday 9/22/15. Left samples in 10 uL of water in 4degC overnight.
  • Qubit of samples for phylogeographic study. Made list of samples for GBS library.
      1. BC1_12: 25.2
      2. BC1_8: 6.44 (8)
      3. BC2_7: 10.6
      4. BC2_13: 11.5
      5. BC2_9: 13 (8)
      6. BC3_13: 108
      7. BC3_12: 15 (8)
      8. BC4_2: 8.84
      9. BC4_17: 11.4 (8)
      10. CA1_18: 24.4
      11. CA1_1: 36
      12. CA1_2: 17.4 (8)
      13. CA2_9: 43.8
      14. CA2_12: 18.8 (5)
      15. CA3_6: 62.3 (5)
      16. CA4_1: 5.1
      17. CA4_7: 19.8 (8)
      18. CA5_10: 48.8 (8)
      19. CA6_8: 5.14
      20. CA6_15: 25.2 (7)
      21. CA7_11: 7.4
      22. CA7_16: 19.1
      23. CA7_15: 14.9 (8)
      24. OR1_ 1: 7.66
      25. OR1_5: 29.2 (7)
      26. OR2_6: 12.9
      27. OR2_12: 15.8
      28. OR2_15: 18.2 (7)
      29. OR3_15: 9.24
      30. OR3_20: 13.7 (7)
      31. WA10_16: 25.4
      32. WA10_13: 9.52 (8)
      33. WA11_10: 18.7
      34. WA11_22: 17.4
      35. WA11_4: 11.7
      36. WA11_17: 4.78 (8)
      37. WA12_15: 24.4
      38. WA12_11: 4.18  (8)
      39. WA13_12: 7.2
      40. WA13_5: 4.4
      41. WA13_15: 45.1 (9)
      42. WA9_2: 11.4 (7)
      43. WA1_16: 62 (8)
      44. Conch_1: 12.4
      45. conch_2: 12.3
      46. conch_4: 9.65
      47. CA5_12: 21.8 (repeat with 2)
      48. CA5_10 repeat within

Friday 9/11/15

Thought I’d be switching my online notebook over to the Open Notebook Science network, but when trying to write this post I found that I could not add a new category. Will try to figure that out, but until then will keep posting here.

Took my samples and reagents from the freezer in my lab on campus to the Pritzker DNA Lab at the Field Museum, where I do the bulk of my molecular work. Spent the day alternating between writing my Doctoral Dissertation Improvement Grant (DDIG) and some lab work.

Common Garden Experiment

  • Extracted DNA from 14 broodstock samples using the EZNA Mollusc kit (protocol here). Left to digest for 2.5 hours.
    1. SS2_6
    2. SS2_7
    3. SS2_8
    4. HC1_6
    5. HC1_7
    6. HC1_8
    7. HC1_9
    8. HC1_10
    9. NF1_3
    10. NF1_4
    11. NF1_5
    12. NF1_6
    13. NF1_7
    14. NF1_8
  • Ran out a gel from the DNA extractions of larvae samples done on 8/11/12 at Manchester.
    • 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
    • The computer hooked up to the UV camera wouldn’t recognize a USB drive, so I just have a phone picture of a printed out picture for now. 12 and 13 are cut off, but they did not show up on gel. For gels of DNA extracts, I give a ranking from 1 (did not work at all) to 5 (bright band of high molecular weight DNA). These are put of the Sample Master Sheet.
      • gel_9_11_15
        1. 3.5
        2. 4
        3. 4
        4. 1 (rerun)
        5. 4
        6. 4.5
        7. 3.5
        8. 3 (low)
        9. 5
        10. 4
        11. 1 (rerun)
        12. 1 (rerun)
        13. 1 (rerun)
        14. 1 (rerun)
        15. 5
        16. 3 (degrad)
        17. 2.5 (degrad)
        18. 2 (degrad)
        19. 5
        20. 1 (rerun)
        21. 4.5
        22. 3 (degrad)
        23. 2.5 (low)
        24. 5
  • Playing around with data!
    • Looked at larvae release from each population across time. Did this partially out of burning curiosity and because it would be nice to have some preliminary data to put in my DDIG proposal.
    • First, I had to edit the Google Sheet to include zeros for days when a population released no larvae and to have total counts across families. Then in R:
    •  > larvae = read.csv("Larval counts - Day 1 (1).csv", header = TRUE) 
      > names(larvae)

      [1] “Date” “Population” “Family” “Tank.added.to” “Volume.of.tripour..mL.” “Vol.of.drop.counts” “Ethanol.used.”
      [8] “Live.Count.1” “Live.Count.2” “Live.Count.3” “Live.Count.4” “Total.Live.Larvae” “X…” “Notes”
      [15] “Dead.count..1” “Dead..2” “Dead..3” “Dead..4” “Total.Dead” “Total.Larvae” “Total.by.date”

 > pop_Total_Date_na <- na.omit(pop_Total_Date)
> ggplot(data=pop_Total_Date_na, aes(x=Date, y=Total.by.date, \
group=Population, colour=Population)) + geom_line() + geom_point()

Day1Larvae_date

  • Interestingly, the South Sound population produced more larvae earlier. This mirrors the reciprocal transplant experiment, where SS oysters reached their maximum percentage of brooding females sooner at two of the 4 sites.

Oly Population Structure

  • In addition to making libraries for samples from the common garden, I need to start getting DNA ready for one more sequencing run for my project looking at rangewide population structure in Olympia oysters. Did 10 extractions concurrently with the common garden extractions.
  1. WA1_16
  2. WA1_14
  3. BC1_19
  4. BC1_18
  5. CA6_16
  6. CA6_17
  7. CA6_18
  8. CA7_13
  9. OR3_7
  10. OR3_20

Wednesday 8/19/15 and Thursday 8/20/15

Wednesday 8/19/15

There was a silo making party at the hatchery today for the rock scallop project. With little animal husbandry to do, I decided to do a DNA extraction and help out making silos while the tissue was digesting.

Tile Culling

  • HCA_7: 33 removed
  • NFA_1: 123
  • NF_A_2: 134
  • NF_A_3: 102
  • NF_A_4: 32

Cultch Set

NF_A + NF_New -> NF> 450_B; NF_New

DNA Extraction

Took tissue out of RNALater onto sterile weigh boat and cut off ~30 mg of tissue with scalpel (have previously weighed out chunks of adductor muscle tissue to help eyeball this amount). Replaced rest of tissue in RNALater. Wiped scalpel and forceps with 85% ethanol and sterilized over benchtop bunsen burner in between samples. Otherwise, followed EZNA protocol as described in a previous post.

  1. SS2_1
  2. SS2_2
  3. SS2_3
  4. SS2_4
  5. SS2_5
  6. HC1_1
  7. HC1_2
  8. HC1_3
  9. HC1_4
  10. HC1_5
  11. NF1_1
  12. NF1_2

Thursday 8/20/15

Tile Set B

  • bleached line
  • cleaned tanks
  • did counts for HC_SetB and NF_SetB

Tile Set A

Had a discussion with Ryan on the ferry about outplanting my tiles. He recommended transitioning the setters slowly to ambient temperature (they are still on heated at 18degC). My room at the hatchery is not really set up for this, so Alice helped me set up one of the large tanks outside that has a heated and an ambient spigot. I tried to just set the tiles on the large 400um mesh screens, but they did not all fit so I just randomized the tiles among 3 poultry wire setups (the same that were in the setting system tanks). The spigots are at one end of the tank and the outflow is at the other, promoting water flow. Food is splashed in by hand and then added in through a dropper. Some of the outflow is actually pumped back in to the side of the tank with the tiles to reduce algae waste.

Tile culling

  • NFA_5: 29 removed
  • SSA_3: 15
  • SSA_5: 61
  • SSA_2: 10
  • SSA_6: 13

Cultch Set

  • NF >1000 -> NF >1600; NF> 1000
  • NF > 450 ->  NF > 1000; NF > 450
  • HC > 1000 -> HC > 1600; HC > 1000
  • HC> 450  ->  HC > 1000; HC > 450