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.

Advertisements

10/29/15

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

  1. HC2_15
  2. HC2_16
  3. HC2_17
  4. HC2_18
  5. HC2_19
  6. HC2_20
  7. HC3_1
  8. HC3_2
  9. HC3_3
  10. HC3_4
  11. HC3_5
  12. HC3_6
  13. HC3_7
  14. HC3_8
  15. HC3_9
  16. HC3_10
  17. HC3_11
  18. HC3_12
  19. HC3_13
  20. HC3_14
  21. HC3_15
  22. HC3_16
  23. HC3_17
  24. HC3_18

Qubit of extractions done on 10/21/15 and 10/28/15. Updated the sample sheet with concentrations.

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.

Monday 10/19/15

Set-up a MiSeq run with the 2b-rad library (concentration of .815 ng/uL) finished on Friday 10/16/15. I’m using the MiSeq in the Pritzker DNA Lab at the Field Museum and a v3 reagent kit. To prepare the library for sequencing, I followed Illumina’s instructions. These instructions 1st call for a 4nM library, which I prepared based off these instructions: CALCULATIONSANDCONVERSIONSINPREPARATIONFORIlluminaMiseqRUN. The fragments should be approximately 176bp, so I used the following formula to get the nM conversion factor: 10^6/660/N (where N is the size of the fragment in basepairs). Taken from this website. This formula gave 8.6 which I rounded to 8. Therefore, to dilute my library:

  • 8nM*.815ng/uL = 6.5nM
  • Dilution: 6.5nM/4nM = 1.625 -> 1 uL sample + .625 water
  • Dilution x 5: 5 uL sample + 3.125 water = 8.125 total volume.

While preparing the library, Illumina gives options for the PhiX spike-in and the concentration to run on the flow cell. I chose a 2% PhiX spike in and 10pM as previous users of this MiSeq have reported overclustering at 11pM.

Friday 10/16/15

Common Garden 2b-RAD

Gel extracted 2b-rad PCR samples from Wednesday 10/14/15 using the Qiagen Gel Extraction Kit. Incubated 40 uL of elution buffer on the column for 5 minutes. Decided that going forward I would run PCR product out on a low melt gel and use gelase instead of the kit to save time/money.

Quantified gel extracted samples with High Sensitivity Qubit. Multiplied 37 uL by the lowest concentration to get ng per sample to add to pool, then calculated how many uL of each sample to add to pool. 37 * .373 = 13.8 ng.

Population Sample ng/uL after gel Vol to add to pool (uL)
Oyster Bay BS_2_6 0.907 15.21499449
Oyster Bay BS_2_7 0.388 35.56701031
Oyster Bay BS_2_8 0.661 20.8774584
Hood Canal BS_1_5 0.731 18.87824897
Hood Canal BS_1_6 1.45 9.517241379
Hood Canal BS_1_7 0.853 16.17819461
Hood Canal BS_1_8 0.96 14.375
Fidalgo BS_1_4 0.873 15.80756014
Fidalgo BS_1_5 0.373 36.99731903
Fidalgo BS_1_6 1.07 12.89719626

GBS Population Structure

Made new annealed adaptors for GBS as the current stock was over 2 years old. Followed Buckler lab protocol. buckler_lab_genotyping_by_sequencing_protocol_20110808

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.