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.

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 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

Tuesday 10/27/15

Finished re-equilibrating the columns that were left in 1M HCL last night. I rinsed them 5 times with water by adding 700 uL of NFW to the column and centrifuging for 1 minute at 10,000g. Then I rinsed them 3 times with TE buffer. Then, following the optional column equilibration step in the EZNA kit manual I added 100 uL 3M NaOH at centrifuged at max speed for 1 minute.

Using these columns, I finished the DNA extraction that was started yesterday evening following the EZNA protocol. I then ran out the extraction on the DNeasy columns from 10/26 and the EZNA extractions with regenerated columns on a gel.

Gel pic of DNA extracted using DNeasy kit.

Gel pic of DNA extracted using DNeasy kit.

  1. SS1_1B: 18.2
  2. SS2_9B: 61.6
  3. SS2_10B: 14.6
  4. SS2_11B: 21.8
  5. SS2_12B: 94
  6. SS2_13B: 24.4
  7. SS2_14B: 276
  8. SS2_16B: 16.2
  9. SS2_17B: 33.6
  10. SS2_18B: 16.7
  11. SS2_19B: 10.8
  12. SS3_3B: 94.8
  13. SS3_4B: 41.2
  14. SS3_7B: 19.9
  15. SS3_8B: 24.6
  16. SS3_9B: 41.2
  17. SS3_10B: 20.8
  18. SS3_11B: 27.2
  19. SS3_13B: 19.7
  20. SS3_14B: 88
  21. SS3_15B: 123
  22. SS3_16B: 126
  23. SS4_1: 19.2
  24. SS4_4: 15.5
Gel of DNA extracted using the EZNA kit and regenerated columns. Tissue was left in lysis buffer overnight.

Gel of DNA extracted with EZNA kit using regenerated columns. Tissue was left in lysis buffer overnight.

  1. SS2_15B: 23.5
  2. SS3_12B: 81.2
  3. SS3_18B: 7.92
  4. SS3_19: 13.9
  5. SS3_20: 48
  6. SS3_21: 19.5
  7. SS4_2: 40
  8. SS4_3: 78.8
  9. SS4_5: 24.7
  10. SS4_6: 26.8
  11. SS4_7: 50.8
  12. SS4_8: 20.4
  13. SS4_9: 11.8
  14. SS4_10: 11.3
  15. SS4_11: 18.2
  16. SS4_12: 35.4
  17. SS4_13: 31.4
  18. SS4_!4: 30.3
  19. SS4_15: 18.3
  20. SS4_16: 26.6
  21. SS4_17: 24.7
  22. SS4_18: 38.7

As you can see, neither of them look very good. With the extraction using regenerated columns, I’m not sure if it was the columns or the fact that they were left overnight. At least the SS2 samples were all repeats, but the SS4 samples will likely have to be re-extracted. Just to be safe I won’t be leaving samples digesting overnight anymore or  trying to regenerate columns.

The new EZNA kit came in in the afternoon, so I did a set of extractions using that kit and did not leave them overnight.

  1. SS4_19
  2. SS4_20
  3. SS5_1
  4. SS5_2
  5. SS5_3
  6. SS5_4
  7. SS5_5
  8. SS5_6
  9. SS5_7
  10. SS5_8
  11. SS5_9
  12. SS5_10
  13. SS5_11
  14. SS5_12
  15. SS5_13
  16. SS5_14
  17. SS5_15
  18. SS5_16
  19. SS5_17
  20. SS5_18
  21. SS5_19

Monday 10/26/15

DNA Extractions for Common Garden Project

I ran out of columns in the EZNA Mollusc kit and the new one I ordered hadn’t come in yet. Since I’m trying to finish all of the South Sound and Hood Canal extractions before I leave on Sunday, I decided to do one set of extractions with the Qiagen DNeasy Kit. I’ve used this kit successfully for my population structure project. Annoyingly, I grabbed the wrong set of samples and ended up re-extracting ones I had done previously. I guess the more DNA, the better. These are notated with a B in the sample sheet. Used Qubit to get DNA concentrations.

  1. SS1_1: 18.2
  2. SS2_9: 61.6
  3. SS2_10: 14.6
  4. SS2_11: 21.8
  5. SS2_12: 94
  6. SS2_13: 24.4
  7. SS2_14: 276
  8. SS2_16: 16.2
  9. SS2_17: 33.6
  10. SS2_18: 16.7
  11. SS2_19: 10.8
  12. SS3_3: 94.8
  13. SS3_4: 41.2
  14. SS3_7: 19.9
  15. SS3_8: 24.6
  16. SS3_9: 41.2
  17. SS3_10: 20.8
  18. SS3_11: 27.2
  19. SS3_13: 19.7
  20. SS3_14: 88
  21. SS3_15: 123
  22. SS3_16: 126
  23. SS4_1: 19.2
  24. SS4_4: 15.5

Since today felt like a wasted day, I decided to set up some extractions to finish tomorrow morning. I was also out of Qiagen columns, however, so looked into regenerating some of the EZNA columns. Based on references here and here I added 500 uL of 1 M HCL to used EZNA columns and left them overnight. I made sure to only reuse columns that had previously been used for South Sound extractions just in case there is any DNA carry-over.

  1. SS2_15: 23.5
  2. SS3_12: 81.2
  3. SS3_18: 7.92
  4. SS3_19: 13.9
  5. SS3_20: 48
  6. SS3_21: 19.5
  7. SS4_2: 40
  8. SS4_3: 78.8
  9. SS4_5: 24.7
  10. SS4_6: 26.8
  11. SS4_7: 50.8
  12. SS4_8: 20.4
  13. SS4_9: 11.8
  14. SS4_10: 11.3
  15. SS4_11: 18.2
  16. SS4_12: 35.4
  17. SS4_13: 31.4
  18. SS4_!4: 30.3
  19. SS4_15: 18.3
  20. SS4_16: 26.6
  21. SS4_17: 24.7
  22. SS4_18: 38.7

Left tissue in lysis buffer at 10:30pm at 56degC.

10/23/15

Common Garden Project

DNA extraction from Olympia oyster broodstock

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

Did not find NF2_8…

Olympia Oyster Population Structure Project

I started preparing genotype-by-sequencing libraries using the dried down samples from 10/21/15. Did an ApeKI digestion and ligation of adaptors.

Digestion:

1x 50x
NEB Buffer 3 2.μL 100
ApeKI 1 μL 50
H2O 17 μL 850

Added 20 uL of digestion master mix to each of the 48 wells. Incubated for 2 hours at 75degC then held at 4degC until ligation master mix was ready.

1x 51x
T4 Ligase Buffer 5 μL 255
T4 Ligase 1.6 μL 81.6 μL
H2O 23.4 μL

Added 30 uL of ligation mastermix to each well to give 50 uL total. Incubated at 19degC for 1 hour and 65degC for 20 minutes. Cooled to 0degC and put in freezer.

10/21/15

Common Garden Samples

  • DNA extraction of broodstock tissue. Let digest for 2.5 hours.
    1.  SS2_9
    2. SS2_10
    3. SS2_11
    4. SS2_12
    5. SS2_13
    6. SS2_14
    7. SS2_15
    8. SS2_16
    9. SS2_17
    10. S2_18
    11. S2_19
    12. S3_3
    13. S3_4
    14. S3_7
    15. S3_8
    16. S3_9
    17. S3_10
    18. S3_11
    19. S3_12
    20. S3_13
    21. S3_14
    22. S3_15
    23. S3_16
    24. S3_17

Running low on EZNA kit columns so ordered another kit.

Phylogeography Project

Setup dry down of 200 ng DNA from 48 samples.

BC1_12
BC1_8
BC2_7: 10.6
BC2_13: 11.5
BC2_9: 13 (8)
BC3_13: 108
BC3_12: 15 (8)
BC4_2: 8.84
BC4_17: 11.4 (8)
CA1_18: 24.4
CA1_1: 36
CA1_2: 17.4 (8)
CA2_9: 43.8
CA2_12: 18.8 (5)
CA3_6: 62.3 (5)
CA4_1: 5.1
CA4_7: 19.8 (8)
CA5_10: 48.8 (8)
CA6_8: 5.14
CA6_15: 25.2 (7)
CA7_11: 7.4
CA7_16: 19.1
CA7_15: 14.9 (8)
OR1_ 1: 7.66
OR1_5: 29.2 (7)
OR2_6: 12.9
OR2_12: 15.8
OR2_15: 18.2 (7)
OR3_15: 9.24
OR3_20: 13.7 (7)
WA10_16: 25.4
WA10_13: 9.52 (8)
WA11_10: 18.7
WA11_22: 17.4
WA11_4: 11.7
WA11_17: 4.78 (8)
WA12_15: 24.4
WA12_11: 4.18 (8)
WA13_12: 7.2
WA13_5: 4.4
WA13_15: 45.1 (9)
WA9_2: 11.4 (7)
WA1_16: 62 (8)
Conch_1: 12.4
conch_2: 12.3
conch_4: 9.65
CA5_13: 21.8 (repeat with 2)
CA5_10 (repeat within)

Keeping the calculations of uL to add on page 3 of this Google Sheet.