Monday 11/23/15

Took the tubes with gel slices from Sunday 11/22/15 and centrifuged them at high speed for 1 minute to bring gel in contact with water. Put them in the -80degC freezer for 1.5 hours (Meyer protocol recommends at least 1 hour). Afterwards, centrifuged at maximum speed in the refrigerated centrifuge at 4degC for 15 minutes (Meyer protocol recommends 10-20 minutes). Then pressed gel slice against tube wall and took out between 30-50uL per sample and added then to a PCR plate corresponding to their well positions during the previous 2bRAD steps. I found that if the gel slices were left out of the cold for too long that they were more likely to break up during this step and less supernatant was recovered- in the future will work in batches of 16 and leave the rest in the fridge. Leftover gel slices were put back in the fridge in case they needed to be gel extracted using a kit due to low DNA recovery.

Ran out the gel of wells B3-C4 on a regular gel and cut out the band at around 170bp. Still need to do B4 from the PCR on 11/22/15. Left gel in 40 uL overnight at 4degC.

Gel_2bRAD_Lib1_B3_C4

Gel of samples B-C4 of 2bRAD Library 1

Did the Ligation 2bRAD step on libraries 2 and 3- decided to use 4 as a backup for failed individuals and put that plate in the freezer.

First made new adapters by adding the oligos into 2 separate tubes and leaving at room temp for 10 minutes:

Adaptor 1
150x 
5ILL-NR
7.5
Anti-ILL
7.5
NFW
735
Adaptor 2
150x
3ILL -NR
7.5 uL
Anti-ILL
7.5 uL
NFW
735 uL
Ligation master mix.
1x
149x
2 uM Adaptor 1
5 uL
745
2 uM Adaptor 2
5 uL
745
T4 ligase
1 uL
149
T4 ligase buffer with 10 mM ATP
4 uL
596
NFW
22
3278
10 mM ATP
1
149
Total
38
5662

Added 38 uL to each well.

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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

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.

Tuesday 9/22/15

Lab Work

Set up a test PCR to determine the optimum # of cycles. You want to identify the minimum number of cycles required for a visible product at 166 bp. I chose 4 of the samples and tested them at 12, 17, 22, & 27 cycles as recommend in the protocol.

  • Made 100 uM stock of new ILL-Lib2 adaptor
  • Made 10 uM stocks of ILL-Lib1 and ILL-Lib2 primers
    • 1.5 stock + 13.5 uL NFW
  • Made 1 uM stocks of HT1 and BC1
    • 1uL stock + 99 NFW
1x
17x
NFW
4.6 uL
78.2
10 mM (each) dNTPS
.4
6.8
10 uM ILL-Lib1
.4
6.8
10 uM ILL-Lib2
.4
6.8
1 uM ILL-HT1
1
17
1 uM ILL-BC1
1
17
5X Q5 buffer
4
68
Q5 Taq polymerase
.2
3.4
Added 12 uL master mix to 8 uL ligation.
  1. SS2_3_12x
  2. HC1_1_12x
  3. NF1_1_12x
  4. NF1_2_12x
  5. SS2_3_17x
  6. HC1_1_17x
  7. NF1_1_17x
  8. NF1_2_17x
  9. SS2_3_22x
  10. HC1_1_22x
  11. NF1_1_22x
  12. NF1_2_22x
  13. SS2_3_27x
  14. HC1_1_27x
  15. NF1_1_27x
  16. NF1_2_27x

17 and 18 are 2 of the ligation reactions for comparison.

  • 17: SS_2_3
  • 18: NF_1_1
Programed PCR in thermocyclers 4,5,6. Called 2b12, 2b17, 2b22, and 2b27.
  •  (98°C 5 sec, 60°C 20 sec, 72°C 10 sec) X N cycles

9_22_15

It looks like 22 cycles is best, and worked on all samples so will be using that in the preparatory scale.

Ethanol precipitation

  • Set up an ethanol precipitation of all the broodstock samples I’ve extracted so far, as well as repeats of of the 4 samples used in the test scale PCR:
    • Population Sample Date extracted ng/uL Volume for 1 ug Volume sodium acetate Vol ethanol
      Oyster Bay BS_2_3 19.8 50.50505051 5.05 113.64
      Hood Canal BS_1_1 12.3 81.30081301 8.13 182.93
      Fidalgo BS_1_1 9.13 109.5290252 10.95 246.44
      Fidalgo BS_1_2 17.4 57.47126437 5.75 129.31
      Oyster Bay BS_2_6 23.4 42.73504274 4.27 96.15
      Oyster Bay BS_2_7 16.4 60.97560976 6.10 137.20
      Oyster Bay BS_2_8 16.8 59.52380952 5.95 133.93
      Hood Canal BS_1_5 12.1 82.6446281 8.26 185.95
      Hood Canal BS_1_6 23.5 42.55319149 4.26 95.74
      Hood Canal BS_1_7 14 71.42857143 7.14 160.71
      Hood Canal BS_1_8 22.8 43.85964912 4.39 98.68
      Hood Canal BS_1_9 13.9 71.94244604 7.19 161.87
      Hood Canal BS_1_10 27.7 36.10108303 3.61 81.23
      Fidalgo BS_1_4 8.9 112.3595506 11.24 252.81
      Fidalgo BS_1_5 18.1 55.24861878 5.52 124.31
      Fidalgo BS_1_6 16.1 62.11180124 6.21 139.75
      Fidalgo BS_1_7 35.8 27.93296089 2.79 62.85
      Fidalgo BS_1_8 22.7 44.05286344 4.41 99.12

      Left in ethanol and sodium acetate in -20C overnight.

Monday 9/14/15

Quantified broodstock samples that were extracted 8/19/15 and 9/11/15 with Qubit and ran out gel. Concentrations in ng/uL are below with score of quality based on gel.

9_14_15 Gel

  1. HC1_10: 27.7 ng/uL; 5
  2. NF1_8: 22.7 ng/uL; 3 (some degrad)
  3. HC1_9: 13.9; 3
  4. SS2_8: 16.8; 4.5
  5. HC1_8:22.8; 5
  6. NF1_2: 17.4; 4.5
  7. HC1_6: 23.5; 5
  8. HC1_1: 12.3; 4.5
  9. NF1_5: 18.1; 4.5
  10. SS2_7: 16.4; 5
  11. SS2_4: 10.2; 4
  12. SS2_3: 19.8; 5
  13. SS2_5: 7.83; 5
  14. HC1_5: 12.1; 5
  15. NF1_3: 27.8; 3 (some degrad)
  16. NF1_4: 8.9; 4
  17. HC1_4: 13.8; 4.5
  18. NF1_1: 9.13; 2.5 (low, some degrad)
  19. HC1_7: 14; 4.5
  20. NF1_7: 35.8; 5
  21. NF1_6: 16.1; 5
  22. HC1_2: 15.2; 2 (low)
  23. HC1_3: 43.5; 5
  24. SS2_6: 23.4; 5
  25. SS2_1; 5
  26. SS2_2; 1 (rerun)

Ethanol extraction of 10 broodstock DNA samples to prepare for test 2b-RAD library, following this protocol.

Population Sample Date extracted ng/uL Volume for 1 ug Volume sodium acetate Vol ethanol
Oyster Bay BS_2_3 19.8 50.50505051 5.05 113.64
Oyster Bay BS_2_4 10.2 98.03921569 9.80 220.59
Oyster Bay BS_2_5 7.83 127.7139208 12.77 287.36
Hood Canal BS_1_1 12.3 81.30081301 8.13 182.93
Hood Canal BS_1_2 15.2 65.78947368 6.58 148.03
Hood Canal BS_1_3 43.5 22.98850575 2.30 51.72
Hood Canal BS_1_4 13.8 72.46376812 7.25 163.04
Fidalgo BS_1_1 9.13 109.5290252 10.95 246.44
Fidalgo BS_1_2 17.4 57.47126437 5.75 129.31
Fidalgo BS_1_3 27.8 35.97122302 3.60 80.94

After drying samples for ~10 minutes at 37degC in a Centrivap Concentrator, I added 10 uL of nuclease-free water and left to rehydrate in 4degC fridge.